John Clarkson - Academia.edu (original) (raw)
Papers by John Clarkson
Surface-enhanced Raman scattering (SERS) from citrate-coated colloidal particles suspended in org... more Surface-enhanced Raman scattering (SERS) from citrate-coated colloidal particles suspended in organic solvents is used to study surface reactions in an organic phase. Stable colloids which gave good SERS were produced from ethanol and acetonitrile. SERS was easily discriminated from the bulk solvent Raman scattering. Compounds which are sparingly soluble in water including 2,Z-bipyridine and benzene-1,2,4,5tetracarboxylic acid dianhydride produced good SERS in ethanol. In the latter case, no SERS was observed from an aqueous colloid. Therefore, some form of solvent-dependent molecular recognition may operate. The formation of a bipyridine complex with copper(I1) at the surface in a solvent system in which the ligand and complex are both soluble was observed. The solvent altered surface reactions. In the ethanol colloid, in contrast to the result in aqueous colloid, pyridiw does not replace citrate readily. This work demonstrates the potential for the expansion of the use of colloidal SERS into the field of surface reactions in contact with an organic phase and is related to metal liquid-like films.
Ab&act-In our previous work we have used near infrared Fourier transform Raman spectroscopy to pr... more Ab&act-In our previous work we have used near infrared Fourier transform Raman spectroscopy to provide a reliable, reproducible and quantitative method for in situ reaction monitoring of homo-polymetisation. Model systems such as styrene and methyhnethacrylate were studied. In this present work three commercial monomers have been studied, namely butylacrylate, hydroxylpropylmethacrylate and lauryhnethacrylate. The effects of reaction temperature, monomer additives and 'the presence of oxygen have been elucidated. These reaction variables have been shown to have a significant effect on polymerisaton rate in particular for the butykcrylate system.
Molecular orbital calculations were performed to determine the normal modes and vibrational energ... more Molecular orbital calculations were performed to determine the normal modes and vibrational energies of azobenzene. A semiempirical calculation using the PM3 Hamiltonian and an ab initio calculation carried out at the SCF level using the 6-3 1G basis set gave unsatisfactory predictions especially for vibrations dominated by azo atom displacements. High-level electron correlation ab inifio calculations carried out at the MP2 level improved the fit with experiment but the choice of basis set was found to be critical. When the basis set for the nitrogens of the azo group was changed to the 6-31+G(d) basis set, the calculation gave a satisfactory fit. Normal-mode diagrams and energies are presented, and assignments to experimentally observed vibrational energies of azobenzene are made. The main azo stretch, Y I O , observed at 1440 cm-I, is theoretically predicted at 1450 cm-I. The calculation correctly predicts an increase in frequency in the azo stretch mode upon deuteration of the phenyl rings. Coupling of several phenyl modes with azo vibrations are revealed by the calculation, in agreement with previous assignments of the vibrational spectra of azobenzene and azobenzene derivatives. The calculation indicates why certain in-plane stretching frequencies give rise to relatively intense Raman and resonance Raman scattering. In Raman scattering, the modes giving rise to the strongest scattering involve displacements along the N-N and C-N bonds. The same modes give intense resonance Raman scattering with the stretches along the azo bond providing the greatest intensity.
Resonance Raman scattering and surface-enhanced resonance Raman scattering (SERRS), for the comme... more Resonance Raman scattering and surface-enhanced resonance Raman scattering (SERRS), for the commercial
phenylazonaphthol pigment Ca4B and the structural analogue (CI 15800 in H and and SERRS for Solvent 2O D2O
Yellow 14 (SY14) in H and are presented. The greater signal-to-noise ratio and the advantage of Ñuores- 2O D2O
cence quenching mean that SERRS gives more information than resonance scattering. The spectra conÐrm that CI
15800 is closely related in structure to Ca4B, indicating that the calcium-complexing site in Ca4B is mainly
associated with the keto and carboxyl groups, with the sulphonic acid group playing a minor part. A semiempirical
calculation using the PM3 Hamiltonian is used to assign scattering from CI 15800 and Ca4B. The most intense
Raman scattering, due to in-plane modes with the largest displacements on the phenyl and naphthol rings, is
correctly assigned. Further, the calculation predicts changes due to deuterium exchange of the hydrogen associated
with the hydrazo group which are borne out by experiment. Hence Raman scattering provides a good in situ probe
of the hydrogen-bonded network essential to the properties of these compounds.
UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin b... more UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin– biotin complex and new data from the avidin–2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43.
The binding of sulphate to human serum apo-transferrin has been examined by ultraviolet absorptio... more The binding of sulphate to human serum apo-transferrin has been examined by ultraviolet absorption and ultraviolet resonance Raman difference spectroscopies between pH 6.0 and 9.0. The ultraviolet absorption data reveals a negative feature at 245 nm that increases in magnitude with pH, with an apparent pK a of 7.57, which the Raman difference data reveals to be due to tyrosine. The pK a of this tyrosine is unusually low and is measured at 7.84 by the Raman difference method and is elevated to greater than 9.0 upon addition of sulphate. Previous studies on the N-lobe imply that Tyr 188 is the tyrosine with a low pK a and also that Arg 124 is the primary binding site for the sulphate. The functional relevance may be that with sulphate bound, both carbonate binding and the deprotonation of Tyr will be disfavoured, and as a result so is iron binding. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5,... more An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5, performed with 244 nm excitation, resolves spectral contributions from the tyrosine 33 and tryptophan residues. The UVRR difference spectrum of holo avidin minus apo avidin, reveals details of the changes to avidin's tyrosine 33. No change in the hydrogen bonding state of tyrosine 33 is detected, but a change to a more hydrophobic environment is indicated upon addition of biotin. Tryptophan bands dominate the difference spectrum, with changes corresponding to the movement of tryptophan tortional angles being observed. The increase in tryptophan UVRR bands upon addition of biotin indicates a change to a more hydrophobic environment, highlighting the importance of hydrophobic interactions in the observed strong avidin/biotin binding.
Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin ... more Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A.. Here we show novel binding features of P450 BioIsspecifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K d values in the range 0.1-160 µM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C 3 position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI)-330 (1 mV; for BioI as purified from E. coli (mixed-spin)) 228 (2 mV; for palmitoleic acid-bound BioI)-199 (2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.
It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturatio... more It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27) 5 *) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (G UN 8.7 and 17.9 kJ mol 1 for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27) 5 * in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 10 3 s 1). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 10 2 s 1), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27) 5 *, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.
The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate-CoA (4-... more The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). In order to facilitate electrophilic catalysis, the dehalogenase utilizes a strong polarizing interaction between the active site residues and the benzoyl portion of the substrate [Taylor, K. L., et al. (1995) Biochemistry 34, 13881]. As a result of this interaction, the normal modes of the benzoyl moiety of the bound 4-HBA-CoA undergo a drastic rearrangement as shown by Raman spectroscopy. Here, we present Raman difference spectroscopic data on the product-enzyme complex where the product's benzoyl carbonyl is labeled with 18 O (Cd 18 O) or 13 C (13 CdO) or where the 4-OH group is labeled with 18 O. The data demonstrate that the carbonyl group participates in the most intense normal modes occurring in the Raman spectrum in the 1520-1560 cm-1 region. The substrate analog 4-methylbenzoate-CoA (4-MeBA-CoA) has also been characterized by Raman difference spectroscopy in its free form and bound to the dehalogenase. Upon binding, the 4-MeBA-CoA shows evidence of polarization within the delocalized π-electrons, but to a lesser extent compared to that seen for the product. The use of 4-MeBA-CoA labeled with 18 O at the carbonyl enables us to estimate the degree of electron polarization within the CdO group of the bound 4-MeBA-CoA. The CdO stretching frequency occurs near 1663 cm-1 in non-hydrogen bonding solvents such as CCl 4 , near 1650 cm-1 in aqueous solution, and near 1610 cm-1 in the active site of dehalogenase. From model studies, we can estimate that in the active site the carbonyl group behaves as though it is being polarized by hydrogen bonds approximately 57 kJ mol-1 in strength. Major contributions to this polarization come from hydrogen bonds from the peptide NHs of Gly114 and Phe64. However, an additional contribution, which may account for up to half of the observed shift in ν CdO , originates in the electrostatic field due to the R-helix dipole from residues 121-114. The helix which terminates at Gly114, near the CdO group of the bound benzoyl, provides a dipolar electrostatic component which contributes to the polarization of the CdO bond and to the polarization of the entire benzoyl moiety. The effect of both the helix dipole and the hydrogen bonds on the CdO is a " pull " of electrons onto the carbonyl oxygen, which, in turn, polarizes the electron distribution within the benzoyl π-electron system. The ability of these two factors to polarize the electrons within the benzoyl moiety is increased by the environment about the benzoyl ring; it is surrounded by hydrophobic residues which provide a low-dielectric constant microenvironment. Electron polarization promotes catalysis by reducing electron density at the C4 position of the benzoyl ring, thereby assisting attack by the side chain of Asp145. An FTIR study on the model compound 4-methylbenzoyl S-ethyl thioester, binding to a number of hydrogen bonding donors in CCl 4 , is described and is used to relate the observed shift of the CdO stretching mode of 4-MeBA-CoA in the active site to the hydrogen bonding strength value. Since the shift of the CdO frequency upon binding is due to hydrogen bonding and helix dipole effects, we refer to this bonding strength as the effective hydrogen bonding strength.
Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular m... more Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfi-brillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman micros-copy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.
Temperature and concentration fields have been imaged by Rayleigh scattering in one-dimension on ... more Temperature and concentration fields have been imaged by Rayleigh scattering in one-dimension on a line and by laser induced fluorescence (LIF) of acetone in a 2-D sheet across the diameter of the cylindrical combustion chamber in a rapid compression machine. Experiments were performed in non-reactive and reactive conditions. To investigate the development of combustion, the exothermic decomposition of di-t-butyl peroxide vapor diluted by inert gas was studied. This reaction is characteristic of a conventional thermal ignition. Acetone is a major product. Inert gas mixtures, to study the temperature field in the absence of reaction, were seeded with acetone. The evidence from the experimental results supports the following interpretation. As the piston of the machine moves, it shears gas off the walls of the chamber. This probably creates a roll-up vortex, but more importantly it also collects cool gas from the walls and moves this gas across the cylinder head pushing it forward into a plug at the center. Once the piston stops, there is a stratified component at the center, which is slightly colder than the bulk of the gas, and for a short time afterwards there is very limited mixing by bulk transport of gas from one part to another, because the gas velocity is not very high. Diffusive transport will occur, but the timescale is relatively slow, and the effect hardly shows before 20 to 25 ms after the end of compression. The effect (on the combustion of di-t-butyl peroxide) of this " temperature stratification " at the core of the cylinder is that the reaction develops more slowly in the center than elsewhere. The onset of reaction in a toroidal region is shown unambiguously, and thermal runaway is initiated there. This is demonstrated by LIF measurements through the central plane of the reaction cylinder. From the study of inert mixtures seeded with acetone, it is shown also that the colder core lies just ahead of the piston crown, but it does not reach the central plane until 1 ms after the piston has stopped. Rayleigh scattering on a 1-D line in the central plane proved to be insufficiently sensitive to show the presence of the cooler zone resulting solely from the physical compression. However, the evidence for temperature stratification becomes unequivocal from Rayleigh scattering measurements made in the later stages of the peroxide decomposition. Limits of sensitivity of the scattering technique may be inferred from this. The physical characteristics of the compression are likely to be replicated in other rapid compression machines and are relevant to understanding the spatial development of autoignition in such systems, which has implications also for numerical modeling. There are rather more complicated consequences, than is the case for thermal ignition, for chain-thermal interactions which involve development through the negative temperature-dependent regimes, as occurs in combustion of the alkanes and that of other organic compounds.
The spontaneous decarboxylation of N-carboxy-2-imidazolidone (a model for carboxybiotin) and N(1′... more The spontaneous decarboxylation of N-carboxy-2-imidazolidone (a model for carboxybiotin) and N(1′)carboxybiotin can be followed at high pH by Raman and FTIR spectroscopies. The major bands associated with vibrations of the carboxylate group have been assigned on the basis of quantum mechanical calculations of N-carboxy-2-imidazolidone and N(1′)-carboxy-2-methylbiotin. The carboxylate modes are the asymmetric stretch, coupled to the ureido carbonyl stretch, near 1710 cm -1 , the symmetric stretch near 1340 cm -1 , and the -CO 2scissoring motion near 830 cm -1 . In the case of carboxybiotin, the last two modes are strongly coupled with biotin ring modes. All three carboxylate modes disappear as spontaneous decarboxylation occurs, to be replaced by features attributable to the noncarboxylated ring structures.The HF/6-31G* optimized structure of 2-methylbiotin revealed that the ureido ring portion is essentially planar, in accord with a number of X-ray crystallographic structures of biotin compounds. However, calculations at this level and at the B3LYP/6-31+G(d) level (using density functional theory) predict that the ureido ring in biotin puckers upon carboxylation. Comparison of the structures of carboxybiotin and carboxyimidazolidone, derived at the HF/6-31G* level, indicates that lengths of the ring-nitrogen-to-carboxylate bonds are equal and that the torsional angles about this linkage are very similar. This strong structural similarity provides a rationale for the observation that, at high pH, the spontaneous rates of decarboxylation of these two molecules are very similar.
A state-of-the-art single grating spectrograph for Raman scattering studies within the deep ultra... more A state-of-the-art single grating spectrograph for Raman scattering studies within the deep ultraviolet (DUV) region of the electromagnetic spectrum is discussed. It is based on a high throughput DUV version of a single-stage monochromator originally designed for use in the visible spectral region. Its key components are two identical, newly designed calcium fluoride camera lenses each consisting of five different individual optical elements. The first of these lenses collimates the Raman scattered DUV radiation entering the spectrometer through its entrance slit. The second lens focuses the collimated beam of dispersed Raman scattered DUV radiation emerging from a high-resolution reflection grating onto a charge coupled device (CCD) detector with enhanced DUV sensitivity. A novel high transmission edge filter is used as a blocking device for a sufficient rejection of the Rayleigh line generating a relatively sharp transmittance cutoff at a Stokes Raman wavenumber shift of about ∼450 cm −1 employing 257 nm DUV excitation. Overall, this new spectrograph enables rapid collection of Stokes DUV Raman scattered photons at f /2 wide apertures with sufficiently large signal-to-noise ratios (SNRs) in relatively short acquisition times and with an effective spectral resolution of approximately ∼6 cm −1 . Backscattered Raman spectra of the following chemicals are presented as typical results illustrating the excellent performance characteristics of this new DUV spectrograph for a variety of experimental conditions within different scattering scenarios and for a relatively wide range of commonly used sample preparation techniques: neat cyclohexane, laboratory air, polycrystalline D-glucose, single crystal L-alanine and a dilute aqueous solution of 2 -deoxyadenosine.
A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P45... more A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala 264 . The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at ϳ420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formateligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substratefree enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G.,
Raman and FTIR, spectra of nitrobenzene, nb, and its isotopomers, nb-15 N, nb-13 C 6 and nb-d 5 ;... more Raman and FTIR, spectra of nitrobenzene, nb, and its isotopomers, nb-15 N, nb-13 C 6 and nb-d 5 ; were obtained and the fundamental vibrational modes assigned with the aid of a B3LYP/6-311 þ G** calculation, without the need for scaling of the force constants. The changes in vibrational coupling between the nitro and benzene groups upon certain isotopic substitutions are well modelled by the calculation, which is able to reproduce the isotopic shifts in frequencies for the nitro vibrations, as well as changes in IR intensities. q (J. Clarkson).
Although Raman spectroscopy is capable of providing
We present comprehensive Raman spectra for thin films of Alq 3 , annealed at various temperatures... more We present comprehensive Raman spectra for thin films of Alq 3 , annealed at various temperatures up to 300°C, over the range of 70-1800 cm Ϫ1 . These spectra give strong evidence for structural rearrangement of thin films of Alq 3 upon annealing at temperatures above 200°C. Needle like crystals are observed to grow in the films and confirmed to be comprised of the ␣-Alq 3 polymorph using the low energy Raman spectra. Furthermore, no evidence of the fac isomer or thermal interconversion between the mer and fac isomers of Alq 3 was observed in either the infrared or Raman spectra of the thin films or powder. These results may have implications for the long-term efficiencies of organic light emitting diodes incorporating thin films of Alq 3 .
Theoretical calculations of the structure, internal rotations and vibrations of 2,4,6-trinitrotol... more Theoretical calculations of the structure, internal rotations and vibrations of 2,4,6-trinitrotolune, TNT, in the gas phase were performed at the B3LYP/6-31G* and B3LYP/6-311 þ G** levels of theory. Two genuine energy minimum structures were found. In both structures the 4-nitro group is planar to the phenyl ring, while the 2,6-nitro groups are slightly out of plane with the phenyl ring due to steric interaction with the methyl group. The two structures are related by internal rotations of the methyl and 2, or 6-nitro group. The lowest energy route for interconversion between them is a concerted motion of the methyl group and 2 or 6 nitro group in a 'cog wheel' type of mechanism. The geometry of the low energy structure A is closest to that observed in the crystal structures of TNT, where all three nitro groups are out of plane with the phenyl ring. FTIR and Raman spectra of solid TNT and 13 C, 15 N enriched TNT are presented and assigned with the help of the B3LYP/6-311 þ G** calculations on A. The lower level B3LYP/6-31G* calculation fails to predict the correct vibrational coupling between the nitro and phenyl groups. The B3LYP/6-311 þ G** calculation gives a good prediction of the nitro vibrations and the isotopic shifts observed for TNT isotopomers. q
Governments often tax fuel products to generate revenues to support and stimulate their economies... more Governments often tax fuel products to generate revenues to support and stimulate their economies. They also subsidize the cost of essential fuel products. Fuel taxation and subsidization practices are both subject to fraud. Oil marketing companies also suffer from fuel fraud with loss of legitimate sales and additional quality and liability issues. The use of an advanced marking system to identify and control fraud has been shown to be effective in controlling illegal activity. DeCipher has developed surface enhanced Raman scattering (SERS) spectroscopy as its lead technology for measuring markers in fuel to identify and control malpractice. SERS has many advantages that make it highly suitable for this purpose. The SERS instruments are portable and can be used to monitor fuel at any point in the supply chain. SERS shows high specificity for the marker, with no false positives. Multiple markers can also be detected in a single SERS analysis allowing, for example, specific regional monitoring of fuel. The SERS analysis from fuel is also quick, clear and decisive, with a measurement time of less than 5 minutes. We will present results highlighting our development of the use of a highly stable silver colloid as a SERS substrate to measure the markers at ppb levels. Preliminary results from the use of a solid state SERS substrate to measure fuel markers will also be presented.
Surface-enhanced Raman scattering (SERS) from citrate-coated colloidal particles suspended in org... more Surface-enhanced Raman scattering (SERS) from citrate-coated colloidal particles suspended in organic solvents is used to study surface reactions in an organic phase. Stable colloids which gave good SERS were produced from ethanol and acetonitrile. SERS was easily discriminated from the bulk solvent Raman scattering. Compounds which are sparingly soluble in water including 2,Z-bipyridine and benzene-1,2,4,5tetracarboxylic acid dianhydride produced good SERS in ethanol. In the latter case, no SERS was observed from an aqueous colloid. Therefore, some form of solvent-dependent molecular recognition may operate. The formation of a bipyridine complex with copper(I1) at the surface in a solvent system in which the ligand and complex are both soluble was observed. The solvent altered surface reactions. In the ethanol colloid, in contrast to the result in aqueous colloid, pyridiw does not replace citrate readily. This work demonstrates the potential for the expansion of the use of colloidal SERS into the field of surface reactions in contact with an organic phase and is related to metal liquid-like films.
Ab&act-In our previous work we have used near infrared Fourier transform Raman spectroscopy to pr... more Ab&act-In our previous work we have used near infrared Fourier transform Raman spectroscopy to provide a reliable, reproducible and quantitative method for in situ reaction monitoring of homo-polymetisation. Model systems such as styrene and methyhnethacrylate were studied. In this present work three commercial monomers have been studied, namely butylacrylate, hydroxylpropylmethacrylate and lauryhnethacrylate. The effects of reaction temperature, monomer additives and 'the presence of oxygen have been elucidated. These reaction variables have been shown to have a significant effect on polymerisaton rate in particular for the butykcrylate system.
Molecular orbital calculations were performed to determine the normal modes and vibrational energ... more Molecular orbital calculations were performed to determine the normal modes and vibrational energies of azobenzene. A semiempirical calculation using the PM3 Hamiltonian and an ab initio calculation carried out at the SCF level using the 6-3 1G basis set gave unsatisfactory predictions especially for vibrations dominated by azo atom displacements. High-level electron correlation ab inifio calculations carried out at the MP2 level improved the fit with experiment but the choice of basis set was found to be critical. When the basis set for the nitrogens of the azo group was changed to the 6-31+G(d) basis set, the calculation gave a satisfactory fit. Normal-mode diagrams and energies are presented, and assignments to experimentally observed vibrational energies of azobenzene are made. The main azo stretch, Y I O , observed at 1440 cm-I, is theoretically predicted at 1450 cm-I. The calculation correctly predicts an increase in frequency in the azo stretch mode upon deuteration of the phenyl rings. Coupling of several phenyl modes with azo vibrations are revealed by the calculation, in agreement with previous assignments of the vibrational spectra of azobenzene and azobenzene derivatives. The calculation indicates why certain in-plane stretching frequencies give rise to relatively intense Raman and resonance Raman scattering. In Raman scattering, the modes giving rise to the strongest scattering involve displacements along the N-N and C-N bonds. The same modes give intense resonance Raman scattering with the stretches along the azo bond providing the greatest intensity.
Resonance Raman scattering and surface-enhanced resonance Raman scattering (SERRS), for the comme... more Resonance Raman scattering and surface-enhanced resonance Raman scattering (SERRS), for the commercial
phenylazonaphthol pigment Ca4B and the structural analogue (CI 15800 in H and and SERRS for Solvent 2O D2O
Yellow 14 (SY14) in H and are presented. The greater signal-to-noise ratio and the advantage of Ñuores- 2O D2O
cence quenching mean that SERRS gives more information than resonance scattering. The spectra conÐrm that CI
15800 is closely related in structure to Ca4B, indicating that the calcium-complexing site in Ca4B is mainly
associated with the keto and carboxyl groups, with the sulphonic acid group playing a minor part. A semiempirical
calculation using the PM3 Hamiltonian is used to assign scattering from CI 15800 and Ca4B. The most intense
Raman scattering, due to in-plane modes with the largest displacements on the phenyl and naphthol rings, is
correctly assigned. Further, the calculation predicts changes due to deuterium exchange of the hydrogen associated
with the hydrazo group which are borne out by experiment. Hence Raman scattering provides a good in situ probe
of the hydrogen-bonded network essential to the properties of these compounds.
UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin b... more UV resonance Raman (UVRR) spectroscopy is used to study the binding of biotin and 2-iminobiotin by streptavidin, and the results are compared to those previously obtained from the avidin– biotin complex and new data from the avidin–2-iminobiotin complex. UVRR difference spectroscopy using 244-nm excitation reveals changes to the tyrosine (Tyr) and tryptophan (Trp) residues of both proteins upon complex formation. Avidin has four Trp and only one Tyr residue, while streptavidin has eight Trp and six Tyr residues. The spectral changes observed in streptavidin upon the addition of biotin are similar to those observed for avidin. However, the intensity enhancements observed for the streptavidin Trp Raman bands are less than those observed with avidin. The changes observed in the streptavidin Tyr bands are similar to those observed for avidin and are assigned exclusively to the binding site Tyr 43 residue. The Trp and Tyr band changes are due to the exclusion of water and addition of biotin, resulting in a more hydrophobic environment for the binding site residues. The addition of 2-iminobiotin results in spectral changes to both the streptavidin and avidin Trp bands that are very similar to those observed upon the addition of biotin in each protein. The changes to the Tyr bands are very different than those observed with the addition of biotin, and similar spectral changes are observed in both streptavidin and avidin. This is attributable to hydrogen bond changes to the binding site Tyr residue in each protein, and the similar Tyr difference features in both proteins supports the exclusive assignment of the streptavidin Tyr difference features to the binding site Tyr 43.
The binding of sulphate to human serum apo-transferrin has been examined by ultraviolet absorptio... more The binding of sulphate to human serum apo-transferrin has been examined by ultraviolet absorption and ultraviolet resonance Raman difference spectroscopies between pH 6.0 and 9.0. The ultraviolet absorption data reveals a negative feature at 245 nm that increases in magnitude with pH, with an apparent pK a of 7.57, which the Raman difference data reveals to be due to tyrosine. The pK a of this tyrosine is unusually low and is measured at 7.84 by the Raman difference method and is elevated to greater than 9.0 upon addition of sulphate. Previous studies on the N-lobe imply that Tyr 188 is the tyrosine with a low pK a and also that Arg 124 is the primary binding site for the sulphate. The functional relevance may be that with sulphate bound, both carbonate binding and the deprotonation of Tyr will be disfavoured, and as a result so is iron binding. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5,... more An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5, performed with 244 nm excitation, resolves spectral contributions from the tyrosine 33 and tryptophan residues. The UVRR difference spectrum of holo avidin minus apo avidin, reveals details of the changes to avidin's tyrosine 33. No change in the hydrogen bonding state of tyrosine 33 is detected, but a change to a more hydrophobic environment is indicated upon addition of biotin. Tryptophan bands dominate the difference spectrum, with changes corresponding to the movement of tryptophan tortional angles being observed. The increase in tryptophan UVRR bands upon addition of biotin indicates a change to a more hydrophobic environment, highlighting the importance of hydrophobic interactions in the observed strong avidin/biotin binding.
Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin ... more Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A.. Here we show novel binding features of P450 BioIsspecifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K d values in the range 0.1-160 µM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C 3 position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI)-330 (1 mV; for BioI as purified from E. coli (mixed-spin)) 228 (2 mV; for palmitoleic acid-bound BioI)-199 (2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.
It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturatio... more It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27) 5 *) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (G UN 8.7 and 17.9 kJ mol 1 for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27) 5 * in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 10 3 s 1). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 10 2 s 1), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27) 5 *, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.
The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate-CoA (4-... more The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). In order to facilitate electrophilic catalysis, the dehalogenase utilizes a strong polarizing interaction between the active site residues and the benzoyl portion of the substrate [Taylor, K. L., et al. (1995) Biochemistry 34, 13881]. As a result of this interaction, the normal modes of the benzoyl moiety of the bound 4-HBA-CoA undergo a drastic rearrangement as shown by Raman spectroscopy. Here, we present Raman difference spectroscopic data on the product-enzyme complex where the product's benzoyl carbonyl is labeled with 18 O (Cd 18 O) or 13 C (13 CdO) or where the 4-OH group is labeled with 18 O. The data demonstrate that the carbonyl group participates in the most intense normal modes occurring in the Raman spectrum in the 1520-1560 cm-1 region. The substrate analog 4-methylbenzoate-CoA (4-MeBA-CoA) has also been characterized by Raman difference spectroscopy in its free form and bound to the dehalogenase. Upon binding, the 4-MeBA-CoA shows evidence of polarization within the delocalized π-electrons, but to a lesser extent compared to that seen for the product. The use of 4-MeBA-CoA labeled with 18 O at the carbonyl enables us to estimate the degree of electron polarization within the CdO group of the bound 4-MeBA-CoA. The CdO stretching frequency occurs near 1663 cm-1 in non-hydrogen bonding solvents such as CCl 4 , near 1650 cm-1 in aqueous solution, and near 1610 cm-1 in the active site of dehalogenase. From model studies, we can estimate that in the active site the carbonyl group behaves as though it is being polarized by hydrogen bonds approximately 57 kJ mol-1 in strength. Major contributions to this polarization come from hydrogen bonds from the peptide NHs of Gly114 and Phe64. However, an additional contribution, which may account for up to half of the observed shift in ν CdO , originates in the electrostatic field due to the R-helix dipole from residues 121-114. The helix which terminates at Gly114, near the CdO group of the bound benzoyl, provides a dipolar electrostatic component which contributes to the polarization of the CdO bond and to the polarization of the entire benzoyl moiety. The effect of both the helix dipole and the hydrogen bonds on the CdO is a " pull " of electrons onto the carbonyl oxygen, which, in turn, polarizes the electron distribution within the benzoyl π-electron system. The ability of these two factors to polarize the electrons within the benzoyl moiety is increased by the environment about the benzoyl ring; it is surrounded by hydrophobic residues which provide a low-dielectric constant microenvironment. Electron polarization promotes catalysis by reducing electron density at the C4 position of the benzoyl ring, thereby assisting attack by the side chain of Asp145. An FTIR study on the model compound 4-methylbenzoyl S-ethyl thioester, binding to a number of hydrogen bonding donors in CCl 4 , is described and is used to relate the observed shift of the CdO stretching mode of 4-MeBA-CoA in the active site to the hydrogen bonding strength value. Since the shift of the CdO frequency upon binding is due to hydrogen bonding and helix dipole effects, we refer to this bonding strength as the effective hydrogen bonding strength.
Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular m... more Fibrillin-rich microfibrils are essential elastic structures contained within the extracellular matrix of a wide variety of connective tissues. Microfibrils are characterized as beaded filamentous structures with a variable axial periodicity (average 56 nm in the untensioned state); however, the basis of their elasticity remains unknown. This study used a combination of small angle x-ray scattering and Raman microscopy to investigate further the packing of microfibrils within the intact tissue and to determine the role of molecular reorganization in the elasticity of these microfibrils. The application of relatively small strains produced no overall change in either molecular or macromolecular microfi-brillar structure. In contrast, the application of larger tissue extensions (up to 150%) resulted in a markedly different structure, as observed by both Raman micros-copy and small angle x-ray scattering. These changes occurred at different levels of architecture and are interpreted as ranging from alterations in peptide bond conformation to domain rearrangement. This study demonstrates the importance of molecular elasticity in the mechanical properties of fibrillin-rich microfibrils in the intact tissue.
Temperature and concentration fields have been imaged by Rayleigh scattering in one-dimension on ... more Temperature and concentration fields have been imaged by Rayleigh scattering in one-dimension on a line and by laser induced fluorescence (LIF) of acetone in a 2-D sheet across the diameter of the cylindrical combustion chamber in a rapid compression machine. Experiments were performed in non-reactive and reactive conditions. To investigate the development of combustion, the exothermic decomposition of di-t-butyl peroxide vapor diluted by inert gas was studied. This reaction is characteristic of a conventional thermal ignition. Acetone is a major product. Inert gas mixtures, to study the temperature field in the absence of reaction, were seeded with acetone. The evidence from the experimental results supports the following interpretation. As the piston of the machine moves, it shears gas off the walls of the chamber. This probably creates a roll-up vortex, but more importantly it also collects cool gas from the walls and moves this gas across the cylinder head pushing it forward into a plug at the center. Once the piston stops, there is a stratified component at the center, which is slightly colder than the bulk of the gas, and for a short time afterwards there is very limited mixing by bulk transport of gas from one part to another, because the gas velocity is not very high. Diffusive transport will occur, but the timescale is relatively slow, and the effect hardly shows before 20 to 25 ms after the end of compression. The effect (on the combustion of di-t-butyl peroxide) of this " temperature stratification " at the core of the cylinder is that the reaction develops more slowly in the center than elsewhere. The onset of reaction in a toroidal region is shown unambiguously, and thermal runaway is initiated there. This is demonstrated by LIF measurements through the central plane of the reaction cylinder. From the study of inert mixtures seeded with acetone, it is shown also that the colder core lies just ahead of the piston crown, but it does not reach the central plane until 1 ms after the piston has stopped. Rayleigh scattering on a 1-D line in the central plane proved to be insufficiently sensitive to show the presence of the cooler zone resulting solely from the physical compression. However, the evidence for temperature stratification becomes unequivocal from Rayleigh scattering measurements made in the later stages of the peroxide decomposition. Limits of sensitivity of the scattering technique may be inferred from this. The physical characteristics of the compression are likely to be replicated in other rapid compression machines and are relevant to understanding the spatial development of autoignition in such systems, which has implications also for numerical modeling. There are rather more complicated consequences, than is the case for thermal ignition, for chain-thermal interactions which involve development through the negative temperature-dependent regimes, as occurs in combustion of the alkanes and that of other organic compounds.
The spontaneous decarboxylation of N-carboxy-2-imidazolidone (a model for carboxybiotin) and N(1′... more The spontaneous decarboxylation of N-carboxy-2-imidazolidone (a model for carboxybiotin) and N(1′)carboxybiotin can be followed at high pH by Raman and FTIR spectroscopies. The major bands associated with vibrations of the carboxylate group have been assigned on the basis of quantum mechanical calculations of N-carboxy-2-imidazolidone and N(1′)-carboxy-2-methylbiotin. The carboxylate modes are the asymmetric stretch, coupled to the ureido carbonyl stretch, near 1710 cm -1 , the symmetric stretch near 1340 cm -1 , and the -CO 2scissoring motion near 830 cm -1 . In the case of carboxybiotin, the last two modes are strongly coupled with biotin ring modes. All three carboxylate modes disappear as spontaneous decarboxylation occurs, to be replaced by features attributable to the noncarboxylated ring structures.The HF/6-31G* optimized structure of 2-methylbiotin revealed that the ureido ring portion is essentially planar, in accord with a number of X-ray crystallographic structures of biotin compounds. However, calculations at this level and at the B3LYP/6-31+G(d) level (using density functional theory) predict that the ureido ring in biotin puckers upon carboxylation. Comparison of the structures of carboxybiotin and carboxyimidazolidone, derived at the HF/6-31G* level, indicates that lengths of the ring-nitrogen-to-carboxylate bonds are equal and that the torsional angles about this linkage are very similar. This strong structural similarity provides a rationale for the observation that, at high pH, the spontaneous rates of decarboxylation of these two molecules are very similar.
A state-of-the-art single grating spectrograph for Raman scattering studies within the deep ultra... more A state-of-the-art single grating spectrograph for Raman scattering studies within the deep ultraviolet (DUV) region of the electromagnetic spectrum is discussed. It is based on a high throughput DUV version of a single-stage monochromator originally designed for use in the visible spectral region. Its key components are two identical, newly designed calcium fluoride camera lenses each consisting of five different individual optical elements. The first of these lenses collimates the Raman scattered DUV radiation entering the spectrometer through its entrance slit. The second lens focuses the collimated beam of dispersed Raman scattered DUV radiation emerging from a high-resolution reflection grating onto a charge coupled device (CCD) detector with enhanced DUV sensitivity. A novel high transmission edge filter is used as a blocking device for a sufficient rejection of the Rayleigh line generating a relatively sharp transmittance cutoff at a Stokes Raman wavenumber shift of about ∼450 cm −1 employing 257 nm DUV excitation. Overall, this new spectrograph enables rapid collection of Stokes DUV Raman scattered photons at f /2 wide apertures with sufficiently large signal-to-noise ratios (SNRs) in relatively short acquisition times and with an effective spectral resolution of approximately ∼6 cm −1 . Backscattered Raman spectra of the following chemicals are presented as typical results illustrating the excellent performance characteristics of this new DUV spectrograph for a variety of experimental conditions within different scattering scenarios and for a relatively wide range of commonly used sample preparation techniques: neat cyclohexane, laboratory air, polycrystalline D-glucose, single crystal L-alanine and a dilute aqueous solution of 2 -deoxyadenosine.
A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P45... more A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala 264 . The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at ϳ420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formateligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substratefree enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G.,
Raman and FTIR, spectra of nitrobenzene, nb, and its isotopomers, nb-15 N, nb-13 C 6 and nb-d 5 ;... more Raman and FTIR, spectra of nitrobenzene, nb, and its isotopomers, nb-15 N, nb-13 C 6 and nb-d 5 ; were obtained and the fundamental vibrational modes assigned with the aid of a B3LYP/6-311 þ G** calculation, without the need for scaling of the force constants. The changes in vibrational coupling between the nitro and benzene groups upon certain isotopic substitutions are well modelled by the calculation, which is able to reproduce the isotopic shifts in frequencies for the nitro vibrations, as well as changes in IR intensities. q (J. Clarkson).
Although Raman spectroscopy is capable of providing
We present comprehensive Raman spectra for thin films of Alq 3 , annealed at various temperatures... more We present comprehensive Raman spectra for thin films of Alq 3 , annealed at various temperatures up to 300°C, over the range of 70-1800 cm Ϫ1 . These spectra give strong evidence for structural rearrangement of thin films of Alq 3 upon annealing at temperatures above 200°C. Needle like crystals are observed to grow in the films and confirmed to be comprised of the ␣-Alq 3 polymorph using the low energy Raman spectra. Furthermore, no evidence of the fac isomer or thermal interconversion between the mer and fac isomers of Alq 3 was observed in either the infrared or Raman spectra of the thin films or powder. These results may have implications for the long-term efficiencies of organic light emitting diodes incorporating thin films of Alq 3 .
Theoretical calculations of the structure, internal rotations and vibrations of 2,4,6-trinitrotol... more Theoretical calculations of the structure, internal rotations and vibrations of 2,4,6-trinitrotolune, TNT, in the gas phase were performed at the B3LYP/6-31G* and B3LYP/6-311 þ G** levels of theory. Two genuine energy minimum structures were found. In both structures the 4-nitro group is planar to the phenyl ring, while the 2,6-nitro groups are slightly out of plane with the phenyl ring due to steric interaction with the methyl group. The two structures are related by internal rotations of the methyl and 2, or 6-nitro group. The lowest energy route for interconversion between them is a concerted motion of the methyl group and 2 or 6 nitro group in a 'cog wheel' type of mechanism. The geometry of the low energy structure A is closest to that observed in the crystal structures of TNT, where all three nitro groups are out of plane with the phenyl ring. FTIR and Raman spectra of solid TNT and 13 C, 15 N enriched TNT are presented and assigned with the help of the B3LYP/6-311 þ G** calculations on A. The lower level B3LYP/6-31G* calculation fails to predict the correct vibrational coupling between the nitro and phenyl groups. The B3LYP/6-311 þ G** calculation gives a good prediction of the nitro vibrations and the isotopic shifts observed for TNT isotopomers. q
Governments often tax fuel products to generate revenues to support and stimulate their economies... more Governments often tax fuel products to generate revenues to support and stimulate their economies. They also subsidize the cost of essential fuel products. Fuel taxation and subsidization practices are both subject to fraud. Oil marketing companies also suffer from fuel fraud with loss of legitimate sales and additional quality and liability issues. The use of an advanced marking system to identify and control fraud has been shown to be effective in controlling illegal activity. DeCipher has developed surface enhanced Raman scattering (SERS) spectroscopy as its lead technology for measuring markers in fuel to identify and control malpractice. SERS has many advantages that make it highly suitable for this purpose. The SERS instruments are portable and can be used to monitor fuel at any point in the supply chain. SERS shows high specificity for the marker, with no false positives. Multiple markers can also be detected in a single SERS analysis allowing, for example, specific regional monitoring of fuel. The SERS analysis from fuel is also quick, clear and decisive, with a measurement time of less than 5 minutes. We will present results highlighting our development of the use of a highly stable silver colloid as a SERS substrate to measure the markers at ppb levels. Preliminary results from the use of a solid state SERS substrate to measure fuel markers will also be presented.