Publications by authors named "Cees Gooijer"

57 Publications

Binding of naproxen enantiomers to human serum albumin studied by fluorescence and room-temperature phosphorescence.

Spectrochim Acta A Mol Biomol Spectrosc 2013 Mar 14;105:67-73. Epub 2012 Dec 14.

Biomolecular Analysis and Spectroscopy, LaserLaB, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

The interaction of the enantiomers of the non-steroidal anti-inflammatory drug naproxen (NPX) with human serum albumin (HSA) has been investigated using fluorescence and phosphorescence spectroscopy in the steady-state and time-resolved mode. The absorption, fluorescence excitation, and fluorescence emission spectra of (S)-NPX and (R)-NPX differ in shape in the presence of HSA, indicating that these enantiomers experience a different environment when bound. In solutions containing 0.2M KI, complexation with HSA results in a strongly increased NPX fluorescence intensity and a decreased NPX phosphorescence intensity due to the inhibition of the collisional interaction with the heavy atom iodide. Fluorescence intensity curves obtained upon selective excitation of NPX show 8-fold different slopes for bound and free NPX. No significant difference in the binding constants of (3.8±0.6)×10(5) M(-1) for (S)-NPX and (3.9±0.6)×10(5) M(-1) for (R)-NPX was found. Furthermore, the addition of NPX quenches the phosphorescence of the single tryptophan in HSA (Trp-214) based on Dexter energy transfer. The short-range nature of this mechanism explains the upward curvature of the Stern-Volmer plot observed for HSA: At low concentrations NPX binds to HSA at a distance from Trp-214 and no quenching occurs, whereas at high NPX concentrations the phosphorescence intensity decreases due to dynamic quenching by NPX diffusing into site I from the bulk solution. The dynamic quenching observed in the Stern-Volmer plots based on the longest phosphorescence lifetime indicates an overall binding constant to HSA of about 3×10(5) M(-1) for both enantiomers.
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http://dx.doi.org/10.1016/j.saa.2012.12.007DOI Listing
March 2013

Looking inside catalyst extrudates with time-resolved surface-enhanced Raman spectroscopy (TR-SERS).

Appl Spectrosc 2012 Oct;66(10):1179-85

Debye Institute for Nanomaterials Science, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.

Raman spectroscopy is one of the major characterization methods employed over the last few decades as a nondestructive technique for the study of heterogeneous catalysts and related catalytic reactions. However, the promise of practical applicability on millimeter-sized catalyst bodies, such as extrudates, has not been fulfilled completely. Large fluorescence signals and the highly scattering nature of the extrudates often hamper its practical usage. Different approaches to overcome this problem were examined, including the use of time-resolved Raman spectroscopy (TRRS), spatially offset Raman spectroscopy (SORS), surface-enhanced Raman spectroscopy (SERS), and combinations of these techniques. This paper demonstrates that especially TRRS can provide chemical information at depth within catalyst bodies, overcoming fluorescence background signals and allowing for visualization of analytes at different depths. It also examines the application of time-resolved SERS within catalyst bodies to gain insight into localized activity. With these options a wider applicability of Raman spectroscopy for industrial catalysis research becomes within reach.
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http://dx.doi.org/10.1366/12-06698DOI Listing
October 2012

Stereoselective binding of flurbiprofen enantiomers and their methyl esters to human serum albumin studied by time-resolved phosphorescence.

Chirality 2012 Oct 21;24(10):840-6. Epub 2012 Jun 21.

Department of Biomolecular Analysis and Spectroscopy, LaserLaB, VU University Amsterdam, The Netherlands.

The interaction of the nonsteroidal anti-inflammatory drug flurbiprofen (FBP) with human serum albumin (HSA) hardly influences the fluorescence of the protein's single tryptophan (Trp). Therefore, in addition to fluorescence, heavy atom-induced room-temperature phosphorescence is used to study the stereoselective binding of FBP enantiomers and their methyl esters to HSA. Maximal HSA phosphorescence intensities were obtained at a KI concentration of 0.2 M. The quenching of the Trp phosphorescence by FBP is mainly dynamic and based on Dexter energy transfer. The Stern-Volmer plots based on the phosphorescence lifetimes indicate that (R)-FBP causes a stronger Trp quenching than (S)-FBP. For the methyl esters of FBP, the opposite is observed: (S)-(FBPMe) quenches more than (R)-FBPMe. The Stern-Volmer plots of (R)-FBP and (R)-FBPMe are similar although their high-affinity binding sites are different. The methylation of (S)-FBP causes a large change in its effect on the HSA phosphorescence lifetime. Furthermore, the quenching constants of 3.0 × 10(7) M(-1) s(-1) of the R-enantiomers and 2.5 × 10(7) M(-1) s(-1) for the S-enantiomers are not influenced by the methylation and indicate a stereoselectivity in the accessibility of the HSA Trp to these drugs.
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http://dx.doi.org/10.1002/chir.22080DOI Listing
October 2012

Complementary fluorescence and phosphorescence study of the interaction of brompheniramine with human serum albumin.

J Phys Chem B 2012 Jun 4;116(24):7033-9. Epub 2012 Jun 4.

Department of Biomolecular Analysis and Spectroscopy, LaserLaB, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp300055cDOI Listing
June 2012

pH-dependent complexation of histamine H1 receptor antagonists and human serum albumin studied by UV resonance Raman spectroscopy.

J Phys Chem B 2012 Mar 12;116(12):3808-15. Epub 2012 Mar 12.

Amsterdam LaserLaB, Department of Biomolecular Analysis and Spectroscopy, Vrije Universiteit, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp206409dDOI Listing
March 2012

Noninvasive detection of concealed explosives: depth profiling through opaque plastics by time-resolved Raman spectroscopy.

Anal Chem 2011 Nov 17;83(22):8517-23. Epub 2011 Oct 17.

Biomolecular Analysis and Spectroscopy, LaserLaB, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/ac2018102DOI Listing
November 2011

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography.

Anal Bioanal Chem 2011 Jul 18;400(9):2913-9. Epub 2011 Apr 18.

Department of Biomolecular Analysis & Spectroscopy, LaserLaB, VU University Amsterdam, the Netherlands.

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-β-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of L-MTX and its enantiomeric impurity D-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10(-7) M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of L-MTX. Furthermore, L-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.
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http://dx.doi.org/10.1007/s00216-011-4991-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102850PMC
July 2011

Computational study on the anomalous fluorescence behavior of isoflavones.

J Phys Chem A 2011 Mar 16;115(9):1493-9. Epub 2011 Feb 16.

Amsterdam Center for Multiscale Modeling (ACMM), VU University Amsterdam, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp109059eDOI Listing
March 2011

Direct spectroscopic evidence of 8- and 9-fold coordinated europium(III) species in H(2)O and D(2)O.

J Phys Chem A 2010 Dec 1;114(50):13050-4. Epub 2010 Dec 1.

Institute of Chemistry (Physical Chemistry), University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam, Germany.

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.
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http://dx.doi.org/10.1021/jp1094036DOI Listing
December 2010

Sensitized phosphorescence as detection method for the enantioseparation of bupropion by capillary electrophoresis.

Electrophoresis 2010 Dec;31(23-24):3928-36

Department of Biomolecular Analysis and Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

A new CE detection method was developed for the chiral drug bupropion (a second-generation antidepressant), based on phosphorescence both in the direct and in the sensitized mode using pulsed laser excitation at 266 nm. Electrokinetic chromatography using 5 mM sulfated-α-CD as chiral selector in 25 mM phosphate buffer at pH 3 allowed the separation of bupropion enantiomers with a high chiral resolution (Rs>3). In the sensitized phosphorescence detection mode, excitation energy is transferred from the analyte to an acceptor (1-bromo-4-napthhalenesulfonic acid or biacetyl) followed by time-resolved phosphorescence detection under deoxygenated buffer conditions. Using 2 × 10(-4) M biacetyl as the acceptor an LOD of 2 × 10(-7) M was obtained for each enantiomer, about 40 times better than in the direct mode. Under these separation conditions, no significantly different phosphorescence lifetimes (measured on-line) were obtained for the two bupropion enantiomers. The suitability of the method was demonstrated with the quantification of bupropion in a pharmaceutical formulation and its determination in a spiked urine sample.
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http://dx.doi.org/10.1002/elps.201000352DOI Listing
December 2010

Sensitized enantioselective laser-induced phosphorescence detection in chiral capillary electrophoresis.

Anal Chem 2010 Nov 21;82(22):9410-7. Epub 2010 Oct 21.

Department of Biomolecular Analysis and Spectroscopy, Laser Centre, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/ac101764zDOI Listing
November 2010

Time-resolved spatially offset Raman spectroscopy for depth analysis of diffusely scattering layers.

Analyst 2010 Dec 13;135(12):3255-9. Epub 2010 Oct 13.

Biomolecular Analysis and Spectroscopy, Laser Centre Vrije Universiteit, Amsterdam, The Netherlands.

The objective of this study is to use time-resolved (TR) Raman spectroscopy, spatially offset Raman spectroscopy (SORS), and a combination of these approaches to obtain high quality Raman spectra from materials hidden underneath an opaque layer. Both TR Raman and SORS are advanced techniques that allow for an increased relative selectivity of photons from deeper layers within a sample. Time-resolved detection reduces fluorescence background, and the selectivity for the second layer is improved. By combining this with spatially offset excitation we additionally increased selectivity for deeper layers. Test samples were opaque white polymer blocks of several mm thicknesses. Excitation was carried out with a frequency-doubled Ti:sapphire laser at 460 nm, 3 ps pulse width and 76 MHz repetition rate. Detection was either with a continuous-wave CCD camera or in time-resolved mode using an intensified CCD camera with a 250 ps gate width. The Raman photons were collected in backscatter mode, with or without lateral offset. By measuring the delay of the Raman signal from the second layer (polyethylene terephthalate/PET/Arnite), the net photon migration speeds through Teflon, polythene, Delrin and Nylon were determined. Raman spectra could be obtained from a second layer of PET through Teflon layers up to 7 mm of thickness. The ability to obtain chemical information through layers of diffusely scattering materials has powerful potential for biomedical applications.
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http://dx.doi.org/10.1039/c0an00611dDOI Listing
December 2010

Characterization of hybrid bilayer membranes on silver electrodes as biocompatible SERS substrates to study membrane-protein interactions.

Colloids Surf B Biointerfaces 2010 Nov 14;81(1):212-6. Epub 2010 Jul 14.

Laser Centre, Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.

Hybrid bilayer lipid membranes (HBMs) were built on roughened silver electrodes exhibiting surface-enhanced Raman scattering (SERS) activity. The HBM consisted of a first layer of octadecanethiol (ODT) directly bound to the electrode surface, on which a second layer of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained by self-assembled phospholipid vesicle fusion. The electrochemical properties of the HBM were investigated in situ by cyclic voltammetry (CV), AC voltammetry and electrochemical impedance spectroscopy (EIS). The results indicate that our HBMs are well-formed, and their insulating properties are comparable to those observed for HBM supported by smooth metal substrates. The interaction between the bilayer and the human enzyme cytochrome P450 2D6 (CYP2D6) was investigated. Surface-enhanced resonance Raman scattering (SERRS) measurements in combination with AC and EIS, performed on the same electrode sample, proved that the CYP2D6 is immobilized on the HBM without evident alterations of its active site and without significant perturbations of the bilayer architecture. This study yields novel insights into the properties of HBMs built on roughened surfaces, providing in situ electrochemical characterization of a substrate which is suitable for studying peripheral membrane proteins with SERRS spectroscopy.
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http://dx.doi.org/10.1016/j.colsurfb.2010.07.010DOI Listing
November 2010

The impact of urea-induced unfolding on the redox process of immobilised cytochrome c.

J Biol Inorg Chem 2010 Nov 13;15(8):1233-42. Epub 2010 Jun 13.

Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41125, Modena, Italy.

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.
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http://dx.doi.org/10.1007/s00775-010-0681-7DOI Listing
November 2010

Phosphorescence for sensitive enantioselective detection in chiral capillary electrophoresis.

Anal Chem 2009 Aug;81(15):6226-33

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/ac900750eDOI Listing
August 2009

Excited state processes of 2-butylamino-6-methyl-4-nitropyridine N-oxide in nonpolar solvents. A transient absorption spectroscopy study.

J Phys Chem A 2010 Apr;114(12):4045-50

Laser Centre Vrije Universiteit Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp909468hDOI Listing
April 2010

Effectiveness of charged noncovalent polymer coatings against protein adsorption to silica surfaces studied by evanescent-wave cavity ring-down spectroscopy and capillary electrophoresis.

Anal Chem 2009 Dec;81(24):10172-8

Department of Biomedical Analysis, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1021/ac902128nDOI Listing
December 2009

High-resolution steady-state and time-resolved luminescence studies on the complexes of Eu(III) with aromatic or aliphatic carboxylic acids.

Anal Chim Acta 2009 Oct 9;652(1-2):285-94. Epub 2009 Jun 9.

Institute of Chemistry, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam-Golm, Germany.

Eu(III) luminescence spectroscopy, both in the steady-state and the time-resolved mode, is an appropriate technique to study the properties of complexes between heavy metal ions and humic substances (HS), which play a key role in the distribution of metal species in the environment. Unfortunately, room temperature luminescence spectra of Eu(III) complexes with aromatic and aliphatic carboxylic acids - model compounds of HS binding sites - are too broad to fully exploit their potential analytical information content. It is shown that under cryogenic conditions fluorescence-line-narrowing (FLN) is achieved, and the highly resolved spectra provide detailed information on the complexes. Ten model ligands were investigated. Total luminescence spectra (TLS) were recorded, using the (5)D(0)<--(7)F(0) transition for excitation and the (5)D(0)-->(7)F(1) and (5)D(0)-->(7)F(2) transitions for emission. The energy of the excitation transition depends on the ligand involved and the structure and composition of the complex. For most ligands, discontinuities in the high-resolution TLS indicated that more species, i.e. distinct complex structures, coexisted in the sample. Selective excitation was performed to measure the species-associated luminescence decay times tau. The latter strongly depend on nearby OH oscillators from coordinating water molecules or ligand hydroxyl groups. Furthermore, the asymmetry ratios r, defined as the intensity ratio of the (5)D(0)-->(7)F(2) and (5)D(0)-->(7)F(1) transitions, were calculated and the variation of the excitation energy E(exc) with the splitting of the (7)F(1) triplet (DeltaE) was determined, which yielded the crystal field strength parameter N(nu)(B(2q)), as well as the crystal field parameters B(20) and B(22). An in-depth analysis of the results is presented, providing detailed information on the number of coexisting complexes, their stoichiometry, the number of water molecules in the first coordination sphere and their geometry (symmetry point group).
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http://dx.doi.org/10.1016/j.aca.2009.06.006DOI Listing
October 2009

Picosecond Raman spectroscopy with a fast intensified CCD camera for depth analysis of diffusely scattering media.

Analyst 2009 Jun 20;134(6):1192-7. Epub 2009 Mar 20.

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, The Netherlands.

A spectroscopic depth profiling approach is demonstrated for layers of non-transparent, diffusely scattering materials. The technique is based on the temporal discrimination between Raman photons emitted from the surface and Raman photons originating from a deeper layer. Excitation was carried out with a frequency-doubled, 3 ps Ti:sapphire laser system (398 nm; 76 MHz repetition rate). Time-resolved detection was carried out with an intensified CCD camera that can be gated with a 250 ps gate width. The performance of the system was assessed using 1 mm and 2 mm pathlength cuvettes with powdered PMMA and trans-stilbene (TS) crystals, respectively, or solid white polymer blocks: Arnite (polyethylene terephthalate), Delrin (polyoxymethylene), polythene (polyethylene) and Teflon (polytetrafluoroethylene). These samples were pressed together in different configurations and Raman photons were collected in backscatter mode in order to study the time difference in such media corresponding with several mm of extra net photon migration distance. We also studied the lateral contrast between two different second layers. The results demonstrate that by means of a picosecond laser system and the time discrimination of a gated intensified CCD camera, molecular spectroscopic information can be obtained through a turbid surface layer. In the case of the PMMA/TS two-layer system, time-resolved detection with a 400 ps delay improved the relative intensity of the Raman bands of the second layer with a factor of 124 in comparison with the spectrum recorded with a 100 ps delay (which is more selective for the first layer) and with a factor of 14 in comparison with a non-gated setup. Possible applications will be discussed, as well as advantages/disadvantages over other Raman techniques for diffusely scattering media.
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http://dx.doi.org/10.1039/b821437aDOI Listing
June 2009

Anomalous photophysics of H1 antihistamines in aqueous solution.

J Phys Chem B 2009 May;113(19):6949-57

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp9005907DOI Listing
May 2009

Excited-state double proton transfer in 1H-pyrazolo[3,4-b]quinoline dimers.

J Phys Chem A 2009 May;113(18):5273-9

Analytical Chemistry & Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp8086545DOI Listing
May 2009

Cryogenic fluorescence and absorption spectroscopy studies on monomeric and dimeric species of 2-butylamino-6-methyl-4-nitropyridine N-oxide.

Spectrochim Acta A Mol Biomol Spectrosc 2009 Feb 19;72(1):144-50. Epub 2008 Sep 19.

Analytical Chemistry & Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, De Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands.

2-Butylamino-6-methyl-4-nitropyridine-N-oxide (2B6M) belongs to a group of compounds that can undergo not only excited-state intra-, but also intermolecular proton transfer. The latter of course requires the presence of dimeric species. Previously, we have shown that for 2B6M in aprotic non-polar solvents in the liquid state such dimers play no role. Under these conditions, only one single monomeric species exists, exhibiting anomalous fluorescence behavior, i.e. proton transfer not only starting from the lowest excited electronic singlet state, but also from higher excited states. However, we also noted that under frozen, crystalline matrix conditions more species show up in the spectra. In order to study this multi-species system in more detail, we present absorption and fluorescence experiments on 2B6M, recorded in n-octane at various temperatures between 293 and 5 K. High-resolution spectra are included, not only in fluorescence but also in absorption. We demonstrate that under cryogenic conditions three species can be discerned, two of these providing high-resolution spectra with their main 0-0 lines around 452 and 465 nm, respectively. A detailed vibrational analysis of their emission spectra is included. The third species gives broad-banded spectra, in absorption extending to about 520 nm with its long-wavelength maximum around 460 nm, in emission with a maximum around 535 nm. We tentatively assign the three species to a monomer, a H-bonded dimer and a strongly interacting (pi-pi-stacked) dimer, respectively. We conclude from the excitation spectra that (anomalous) intramolecular proton transfer at higher excited states is still operative under cryogenic conditions. Indications for excited-state intermolecular proton transfer in the stacked dimeric species were not found.
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http://dx.doi.org/10.1016/j.saa.2008.09.003DOI Listing
February 2009

Enantioselective detection of chiral phosphorescent analytes in cyclodextrin complexes.

Talanta 2005 Apr 6;66(3):641-5. Epub 2005 Jan 6.

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre, Vrije Universiteit, De Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands.

Inclusion complexes between camphorquinone (CQ) and cyclodextrins (CDs) in deoxygenated aqueous solutions are shown to exhibit relatively strong room temperature phosphorescence (RTP). Among the various CDs tested, alpha-CD showed the strongest RTP signals. Interestingly, these signals differed significantly for the two enantiomers of CQ; the phosphorescence lifetime of (+)-CQ was about four times longer than that of (-)-CQ, being 352+/-16 and 89+/-6mus, respectively. This enantiomeric selectivity is attributed to a difference in dissociation rates (competing with the radiative emission process) for the diastereoisomeric inclusion complexes dealt with, which have a 2:1 stoichiometry (alpha-CD:CQ:alpha-CD). Time-resolved RTP detection using different delay times enables the determination of the two enantiomers in a mixture without involving a separation technique. The minimum detectable fraction of (+)-CQ in a 2mM sample was 13%.
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http://dx.doi.org/10.1016/j.talanta.2004.12.010DOI Listing
April 2005

Enantioselective room temperature phosphorescence detection of non-phosphorescent analytes based on interaction with beta-cyclodextrin/1-bromonaphthalene complexes.

Talanta 2005 Apr 7;66(3):634-40. Epub 2005 Jan 7.

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre, Vrije Universiteit, De Boelelaan 1083, NL-1081 HV Amsterdam, The Netherlands.

Menthol (MT) induces strong room temperature phosphorescence (RTP) of 1-bromonaphthalene (1BrN) in aqueous beta-cyclodextrin (beta-CD) suspensions, even under non-deoxygenated conditions. Interestingly, (-)-MT and (+)-MT enantiomers give rise to different phosphorescence intensities, the difference being 19+/-3%. It is argued that the signal can be mainly ascribed to the formation of ternary complexes beta-CD/1BrN/MT which show different RTP lifetimes, i.e. 4.28+/-0.06 and 3.71+/-0.06ms for (-)-MT and (+)-MT, respectively. Most probably, the stereochemical structure of (-)-MT provides a better protection of 1BrN against quenching by oxygen than (+)-MT. This interpretation is in line with the observation that under deoxygenated conditions the phosphorescence intensity difference for the two complexes becomes very small, i.e. only about 4%. The lifetime difference under aerated conditions enables the direct determination of the MT stereochemistry. For mixtures, in view of the 0.06ms uncertainty in the lifetime, enantiomeric purity can be determined down to 10%. Furthermore, in the case of MT the concentration of the least abundant enantiomer should be at least 3x10(-4)M, since otherwise complex dissociation would obscure the lifetime difference.
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http://dx.doi.org/10.1016/j.talanta.2004.12.009DOI Listing
April 2005

Achievements in resonance Raman spectroscopy review of a technique with a distinct analytical chemistry potential.

Anal Chim Acta 2008 Jan 9;606(2):119-34. Epub 2007 Nov 9.

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, The Netherlands.

In an extended introduction, key aspects of resonance Raman spectroscopy (RRS) such as enhanced sensitivity and selectivity are briefly discussed in comparison with normal RS. The analytical potential is outlined. Then achievements in different fields of research are highlighted in four sections, with emphasis on recent breakthroughs: (1) The use of visible RRS for analyzing carotenoids in biological matrices, for pigments and dyes as dealt with in art and forensics, and for characterizing carbon nanotubes. (2) The use of RRS in the deep UV (excitation below 260nm) in the bioanalytical and life sciences fields, including nucleic acids, proteins and protein-drug interactions. Metalloproteins can be studied by visible RRS in resonance with their chromophoric absorption. (3) Progress in theoretical calculations of RRS excitation profiles and enhancement factors, which ultimately might facilitate analytical RRS. (4) Instrumental and methodological achievements including fiber-optic UV-RRS, coupling of RRS to liquid chromatography and capillary electrophoresis. Sensitivities can approach the single-molecule level with surface-enhanced RRS or tip-enhanced RRS. Last but not least, promising fluorescence background rejection techniques based on time-gated detection will be presented. This review ends with a concluding section on future expectations for RRS, in particular its potential as an analytical technique.
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http://dx.doi.org/10.1016/j.aca.2007.11.006DOI Listing
January 2008

Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3.

Proteins 2008 Apr;71(1):336-52

Leiden/Amsterdam Centre for Drug Research, Division of Molecular Toxicology, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3.
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http://dx.doi.org/10.1002/prot.21697DOI Listing
April 2008

Active-site structure, binding and redox activity of the heme-thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study.

J Biol Inorg Chem 2008 Jan 26;13(1):85-96. Epub 2007 Sep 26.

Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1083a, 1081 HV Amsterdam, The Netherlands.

Surface-enhance resonance Raman scattering spectra of the heme-thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme's active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme-imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme's ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6.
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http://dx.doi.org/10.1007/s00775-007-0303-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2099460PMC
January 2008

pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes.

Langmuir 2007 Sep 8;23(19):9898-904. Epub 2007 Aug 8.

Laser Centre-Analytical Chemistry and Applied Spectroscopy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/la701751rDOI Listing
September 2007

Fluorescence rejection in resonance Raman spectroscopy using a picosecond-gated intensified charge-coupled device camera.

Appl Spectrosc 2007 Jun;61(6):571-8

Department of Analytical Chemistry and Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, the Netherlands.

A Raman instrument was assembled and tested that rejects typically 98-99% of background fluorescence. Use is made of short (picosecond) laser pulses and time-gated detection in order to record the Raman signals during the pulse while blocking most of the fluorescence. Our approach uses an ultrafast-gated intensified charge-coupled device (ICCD) camera as a simple and straightforward alternative to ps Kerr gating. The fluorescence rejection efficiency depends mainly on the fluorescence lifetime and on the closing speed of the gate (which is about 80 ps in our setup). A formula to calculate this rejection factor is presented. The gated intensifier can be operated at 80 MHz, so high repetition rates and low pulse energies can be used, thus minimizing photodegradation. For excitation we use a frequency-tripled or -doubled Ti : sapphire laser with a pulse width of 3 ps; it should not be shorter in view of the required spectral resolution. Other critical aspects tested include intensifier efficiency as a function of gate width, uniformity of the gate pulse across the spectrum, and spectral resolution in comparison with ungated detection. The total instrumental resolution is 7 cm(-1) in the blue and 15 cm(-1) in the ultraviolet (UV) region. The setup allows one to use resonance Raman spectroscopy (RRS) for extra sensitivity and selectivity, even in the case of strong background fluorescence. Excitation wavelengths in the visible or UV range no longer have to be avoided. The effectiveness of this setup is demonstrated on a test system: pyrene in the presence of toluene fluorescence (lambda(exc) = 257 nm). Furthermore, good time-gated RRS spectra are shown for a strongly fluorescent flavoprotein (lambda(exc) = 405 nm). Advantages and disadvantages of this approach for RRS are discussed.
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http://dx.doi.org/10.1366/000370207781269873DOI Listing
June 2007

Intramolecular proton-transfer processes starting at higher excited states: a fluorescence study on 2-butylamino-6-methyl-4-nitropyridine N-oxide in nonpolar solutions.

J Phys Chem A 2007 Jul 9;111(26):5828-32. Epub 2007 Jun 9.

Analytical Chemistry & Applied Spectroscopy, Laser Centre Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.1021/jp0672813DOI Listing
July 2007
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