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The identification of natural organic pigments is important for the conservation, preservation, and historical interpretation of artwork. Due to the fugitive nature of the natural dye components in pigments, their analysis can be complicated by issues such as low concentration and sample complexity. In addition, these pigments are exceedingly diverse, and often represent complex mixtures which are difficult to analyse without a separation step. A particularly challenging class of dyes are the natural yellow polyphenols (i.e. quercetin, rhamnetin, emodin, etc.) Several techniques have been used successfully for the identification of phenolic compounds in a complex mixture, but the majority of these methods require advanced instrumentation and one or more separation steps. In addition, these methods may lack the sensitivity needed to detect minute amounts of pigment remaining in a faded artwork. As a result, there is a need for innovative methods of analysis which can be applied to the interpretation of artworks containing natural dyes. In this work, cost-effective screen printed electrodes (SPEs) modified with silver nanoparticles (AgNP) were used to amplify the SERS response of phenolic compounds. In particular, application of a voltage to the SERS substrate allows for a fine-tuning of the SERS signal, and in addition was successfully used to separately characterize dye components in two natural yellow lake pigments, Reseda Lake and Stil de Grain. To our knowledge, this work represents the first electrochemical surface-enhanced Raman spectroscopy (EC-SERS) study of polyphenolic dye mixtures, and is the first application of EC-SERS for natural pigment analysis. This work establishes EC-SERS as a useful technique for the identification of complex natural dyes which may find potential use in the cultural heritage realm.

The identification of organic colorants in traditional oil paintings is an important and challenging area of conservation research. Since many organic dyes and pigments are fugitive, the detection ...

Multicolor single-molecule imaging is widely applied to answer questions in biology and materials science. However, most studies rely on spectrally distinct fluorescent probes or time-intensive sequential imaging strategies to multiplex. Here, we introduce blinking-based multiplexing (BBM), a simple approach to differentiate spectrally overlapped emitters based solely on their intrinsic blinking dynamics. The blinking dynamics of hundreds of rhodamine 6G and CdSe/ZnS quantum dots on glass are obtained using the same acquisition settings and analyzed with a change point detection algorithm. Although substantial blinking heterogeneity is observed, the analysis yields a blinking metric with 93.5% classification accuracy. We further show that BBM with up to 96.6% accuracy is achieved by using a deep learning algorithm for classification. This proof-of-concept study demonstrates that a single emitter can be accurately classified based on its intrinsic blinking dynamics and without the need to probe its spectral color.

The aggregation and photodegradation of rhodamine derivatives adsorbed to TiO2 are investigated using diffuse reflectance spectroscopy, steady-state fluorescence, and time-correlated single-photon-counting (TCSPC) measurements. Rhodamine dyes containing substituted amines (i.e., 5-ROX, R101, RB) exhibit an ∼50 nm hypsochromic shift in λmax upon adsorption to TiO2 relative to solution. By examining a rhodamine derivative with primary amines (i.e., R560) as well as control experiments on insulating ZrO2 substrates, we demonstrate that photocatalyzed N-dealkylation is largely responsible for the spectral changes observed upon surface adsorption to TiO2. For R560, which does not undergo N-dealkylation, diffuse reflectance spectra show that mainly monomers and J-aggregates are present on TiO2. Comparative lifetime measurements for R560 on TiO2 and ZrO2 show that the injection yield for R560/TiO2 is increased with dye-loading concentration (i.e., from 0.63 for monomers to ∼0.80 for heavily doped films), indicat...

The blinking and photobleaching dynamics of alizarin (1,2-dihydroxyanthraquinone) and purpurin (1,2,4-trihydroxyanthraquinone) are investigated using single-molecule spectroscopy. The time-dependent emission of alizarin and purpurin on glass under N2 is analyzed using the change point detection (CPD) method to compile on- and off-event distributions. The number of distinct emissive events per molecule is about four times higher for alizarin relative to purpurin, consistent with an excited-state intramolecular proton transfer (ESIPT) process to populate an emissive tautomer state. To elucidate the mechanism for blinking (i.e., switching between on and off events), maximum likelihood estimation (MLE), goodness-of-fit tests based on the Kolmogorov–Smirnov (KS) statistic, and the log-likelihood ratio (LLR) tests are used to establish the best fits to the on- and off-interval probability distributions. For both alizarin and purpurin the on intervals are log-normally distributed, and off intervals are Weibull d...

The intersystem crossing and dispersive electron-transfer dynamics of eosin Y (EY) photosensitizers are probed using single-molecule microscopy. The blinking dynamics of EY on glass are quantified by constructing cumulative distribution functions of emissive ("on") and nonemissive ("off") events. Maximum likelihood estimation (MLE) and goodness-of-fit tests based on the Kolmogorov-Smirnov (KS) statistic are used to establish the best fit to the blinking data and differentiate among competitive photophysical processes. The on-time probability distributions for EY in N2 and air are power-law distributed after ∼1 s, with fit parameters that are significantly modified upon exposure to oxygen. By extending the statistically principled MLE/KS approach to include an onset time for log-normal behavior, we demonstrate that the off-time distribution for EY in N2 is best fit to a combination of exponential and log-normal functions. The corresponding distribution for EY in air is best fit to a log-normal function alone. Furthermore, power law and log-normal distributions are observed for an individual molecule in air, consistent with dynamic fluctuations in the rate constant for dark-state population and depopulation. These observations support the interpretation that dispersive electron transfer (i.e., the Albery model) from the first excited singlet state (S1) of EY to trap states on glass is predominately responsible for blinking in oxic conditions. In anoxic environment, both triplet-state blinking and dispersive electron transfer from S1 and the excited triplet state (T1) contribute to the excited-state dynamics of EY.

The effects of dye structure, driving force of photoinduced electron transfer, and adsorption affinity on the dispersive electron-transfer dynamics of rhodamines on TiO2 films are investigated using single-molecule microscopy. The time-dependent emission (i.e., blinking dynamics) of rhodamine-sensitized TiO2 films are quantified using maximum likelihood estimation (MLE) and quantitative goodness-of-fit tests based on the Kolmogorov–Smirnov (KS) statistic to determine the best fit to the photophysical data. Although the observation of significant p-values (i.e., ranging from 0.16 to 0.53) seems to support the power-law model for the on-time distributions, only a minor subset of the data are actually represented by power laws (i.e., ∼15–20% of events). Instead, the MLE/KS analysis reveals that log-normal distributions, not power laws, most closely represent the entire on-time and off-time distributions for RB, R6G, R123, and 5-ROX on TiO2. Monte Carlo simulations based on the Albery model for dispersive ele...

A novel treatment flowchart approach for surface-enhanced Raman scattering (SERS) is used to identify both blue and yellow organic pigments in a single microscopic sample from a series of reference oil paints as well as an actual 18th century oil painting. In particular, several treatment strategies using acids and solvents are integrated into a specific flowchart designed to enable the minimally invasive identification of unknown blue (i.e., indigo, Prussian blue) and yellow organic (i.e., Reseda lake, Stil de Grain, gamboge) pigments in one sample. We demonstrate the first successful identification of a yellow lake pigment in a historic painting using SERS as well as the utility of our treatment flowchart approach for identifying pigments of varying resonance conditions, surface affinities, and treatment requirements in a single microscopic sample from a historic oil painting.

We demonstrate the first biosensing strategy that relies on quantum dot (QD) fluorescence blinking to report the presence of a target molecule. Unlike other biosensors that utilize QDs, our method does not require the analyte to induce any fluorescence intensity or color changes, making it readily applicable to a wide range of target species. Instead, our approach relies on the understanding that blinking, a single particle phenomenon, is obscured when several QDs lie within the detection volume of a confocal microscope. If QDs are engineered to aggregate when they encounter a particular target molecule, the observation of quasi-continuous emission should indicate its presence. As proof of concept, we programmed DNAs to drive rapid isothermal assembly of QDs in the presence of a target strand (oncogene K-ras). The assemblies, confirmed by various gel techniques, contained multiple QDs and were readily distinguished from free QDs by the absence of blinking.

Dye-doped silica-coated silver nanoparticles (SCNPs) with shell thicknesses ranging from 8.6 ± 1.4 nm to 75.4 ± 4.8 nm are examined at the single-nanoparticle level using correlated transmission electron microscopy (TEM), localized surface plasmon resonance (LSPR), and fluorescence microscopy. The average fluorescence enhancement of 128 individual RB/SCNPs is 2.4 ± 0.2, with individual values ranging from 1.0 to 12.8. For these hot-spot-containing SCNPs, the far-field Rayleigh scattering is not correlated to fluorescence enhancement. Instead, fluorescence enhancement is positively correlated to aggregation state (i.e., the number of hot spots) and dye-metal distance, with ∼19 nm separation producing the largest fluorescence enhancement.

Surface-enhanced Raman scattering (SERS) studies of art represent an attractive way to introduce undergraduate students to concepts in nanoscience, vibrational spectroscopy, and instrumental analysis. Here, we present an undergraduate analytical or physical chemistry laboratory wherein a combination of normal Raman and SERS spectroscopy is used to identify both inorganic and organic fluorescent colorants in an oil painting. On the basis of their experimental observations, students make procedural decisions to adjust acquisition settings and use SERS, thereby enabling the successful identification of unknowns. This laboratory engages undergraduate students by applying what they have learned about quantum mechanics, nanoscience, and spectroscopy to the real-world, problem-solving context of art conservation.

A quality assurance (QA) procedure is developed that enables analysts to effectively test the intrinsic activity of a SERS substrate - silver colloids - before applying them to a precious art sample. SERS measurements of adsorbed citrate or alizarin should not be used to establish substrate fidelity. Instead, we recommend carmine and madder lake pigments as first steps in QA. After colloid activity is established, integrating the QA protocol with sample pretreatment strategies provides for the reproducible identification of unknown colorants in microscopic samples from references and an actual painting.

The distributions of electron-transfer dynamics in dye-sensitized TiO2 films are probed using single-molecule microscopy. The time-dependent emission (i.e., blinking dynamics) of rhodamine 6G (R6G) and rhodamine B (RB) sensitized TiO2 films are quantified by constructing cumulative distribution functions of emissive (“on”) and nonemissive (“off”) events. Maximum likelihood estimation (MLE) methods and quantitative goodness-of-fit tests based on the Kolmogorov–Smirnov (KS) statistics are used to establish the best fit to the photophysical data. The on-time distributions for R6G and RB on TiO2 are fit by power laws, but only for emissive durations that last longer than ∼0.7 s. Furthermore, large variations in the power-law exponents are observed when using least-squares fitting as compared to the combined MLE and KS-test approach. The off-time distributions for molecules on TiO2 and glass are not consistent with power laws and are instead well represented by log-normal distributions. Our observations suppor...

Surface-enhanced Raman scattering (SERS) spectroscopy is increasingly applied to the identification of organic colorants in cultural heritage objects because vibrational fingerprints can be measured from microscopic samples. However, the development of SERS into a reliable, broad-spectrum method for art analysis requires the study of a wide variety of organic and inorganic colorants as well as colorant mixtures in paint. Here, we demonstrate reliable protocols for SERS-based identification of insoluble indigo, Prussian blue (PB), and mixtures thereof in aged painted surfaces. The use of simple salts and acids for sample pretreatment is evaluated. High-quality SERS spectra of PB and indigo are elucidated upon sample pretreatment with H(2)SO(4). In several cases, SERS spectra of the colorants could not be obtained without sample pretreatment. We demonstrate the use of H(2)SO(4) to solubilize PB as well as perform an in situ conversion of insoluble indigo to soluble indigo carmine (IC) on indigo, indigo oil paint, and actual samples from historic painted surfaces. A microscopic H(2)SO(4)-treated sample from the Portrait of Evelyn Byrd produced a SERS spectrum that is consistent with a mixture of PB and IC. To our knowledge, this work represents the first SERS spectrum of indigo in oil paint and the first simultaneous detection of a mixture of blue organic and inorganic colorants in a single art sample using SERS.

A novel spectroscopic approach, correlated surface-enhanced Raman scattering (SERS) and fluorescence microscopy, is used to identify organic materials in two 18th century oil paintings. The vibrational fingerprint of analyte molecules is revealed using SERS, and corresponding fluorescence measurements provide a probe of local environment as well as an inherent capability to verify material identification. Correlated SERS and fluorescence measurements are performed directly on single pigment particles obtained from historic oil paintings with Ag colloids as the enhancing substrate. We demonstrate the first extractionless nonhydrolysis SERS study of oil paint as well as the potential of correlated SERS and fluorescence microscopy studies for the simultaneous identification of organic colorants and binding media in historic oil paintings.

Finite element method calculations were carried out to determine extinction spectra and the electromagnetic (EM) contributions to surface-enhanced Raman spectroscopy (SERS) for 90-nm Au nanoparticle dimers modeled after experimental nanotags. The calculations revealed that the EM properties depend significantly on the junction region, specifically the distance between the nanoparticles for spacings of less than 1 nm. For extinction spectra, spacings below 1 nm lead to maxima that are strongly red-shifted from the 600-nm plasmon maximum associated with an isolated nanoparticle. This result agrees qualitatively well with experimental transmission electron microscopy images and localized surface plasmon resonance spectra that are also presented. The calculations further revealed that spacings below 0.5 nm, and especially a slight fusing of the nanoparticles to give tiny crevices, leads to EM enhancements of 10(10) or greater. Assuming a uniform coating of SERS molecules around both nanoparticles, we determined that regardless of the separation, the highest EM fields always dominate the SERS signal. In addition, we determined that for small separations less than 3% of the molecules always contribute to greater than 90% of the signal.

Large (1 cm3) potassium hydrogen phthalate (KAP) crystals grown in the presence of the fluorescent dye 2′,7′-dichlorofluorescein (DCF) show patterns of luminescence characteristic of selective inte...

The blinking dynamics of single violamine R (VR) molecules embedded in crystals of potassium acid phthalate (KAP) are analyzed using threshold and change-point detection (CPD) methods (Watkins, L. P.; Yang, H. J. Phys. Chem. B 2005, 109, 617). Analysis employing thresholding resulted in power-law distributions of on and off times corresponding to a power-law exponent of ∼2, consistent with a distributed kinetics model for population and depopulation of the nonemissive state. When the same emission time traces were analyzed using the CPD method, a power-law exponent of ∼1.5 is obtained. In addition, multiple emission states are observed using CPD, inconsistent with a simple two-state blinking model, and indicative of spectral diffusion. The role of spectral diffusion in the distributed blinking kinetics of KAP/VR is investigated by spectrally decomposing the emission using a dichroic mirror. Combining the CPD method with this experiment yielded the emission energy, intensity, and temporal duration of blink...

Recent progress in single-molecule spectroscopy of dyed composite materials is reviewed. In particular, single-molecule studies of dyed polymer films and dye inclusion crystals are described that seek to understand local guest–host interactions and environmental heterogeneity. Single-molecule orientational studies of chromophores in polymer films and mixed crystals are discussed with particular relevance to materials and device applications. Studies of single-molecular reorientational dynamics in polymers in the presence of an electric field designed to establish alignment are described. Static orientational measurements in mixed crystals, wherein alignment is a consequence of crystal growth, reveal a surprisingly wide distribution of molecular orientations. Moreover, the complex photophysics of single molecule in anisomorphous mixed crystals are explored and described in the context of environmental heterogeneity and distributed kinetics. The emerging literature regarding dispersed kinetics observed for ...

The intermittent emission or “blinking” of single violamine R (1) and 2‘,7‘-dichlorofluorescein (2) molecules incorporated into single crystals of potassium acid phthalate (KAP) is studied using confocal fluorescence microscopy. Blinking dynamics are quantified in terms of switching rates and on- and off-length probability distributions. Mixed crystals of KAP/1 and KAP/2 consist of photophysical subpopulations with ∼40% and ∼20% exhibiting persistent emission, respectively, and the remainder demonstrating a broad range of blinking behavior that is well described by a power-law distribution. The dependence of the power-law exponent on chromophore, experimental bin time, intensity threshold, and excitation power is examined. The blinking dynamics are also modeled using Monte Carlo simulations on the basis of a three-level electronic system with the rate constants for population and depopulation of the “dark” state being distributed. No correlation between molecular orientation and blinking dynamics is obser...

Single-molecule surface-enhanced Raman spectroscopy (SMSERS) of crystal violet (CV) has been reported since 1997, yet others have offered alternative explanations that do not necessarily imply SMSERS. Recently, the isotopologue approach, a statistically significant method to establish SMSERS, has been implemented for members of the rhodamine dye family. We provide the first demonstration of SMSERS of a triphenylmethane dye using the isotopologue approach. Two isotopologues of CV are employed to create chemically identical yet vibrationally distinct probe molecules. Experimental spectra were compared extensively with computational simulations to assign changes in mode frequencies upon deuteration. More than 90 silver nanoparticle clusters dosed with a 50:50 mixture of CV isotopologues were spectroscopically characterized, and the vibrational signature of only deuterated or undeuterated CV was observed 79 times, demonstrating that the isotopologue approach for proving SMSERS is applicable to both the CV and the rhodamine systems. The use of CV, a minimally fluorescent dye, allowed direct evaluation of enhancement factors (EF), which are reported herein. Through experiment and theory, we show that molecular electronic resonance Raman (RR) and surface-enhanced Raman effects combine synergistically in SMSERS. Excluding RR effects, the EF(SERS) is ∼10(9). Variations and relationships between substrate morphology and optical properties are further characterized by correlated SMSERS-localized surface plasmon resonance (LSPR)-high-resolution transmission electron microscopy (HRTEM) studies. We did not observe SMSERS from individual nanoparticles; further, SMSERS-supporting dimers are heterodimers of two disparately sized particles, with no subnanometer gaps. We present the largest collection to date of HRTEM images of SMSERS-supporting nanoparticle assemblies.

Although plasmonic nanoparticles are widely utilized in spectroscopy and sensing applications, a quantitative structure-function relationship is lacking. In this proceeding, we discuss measurements of single noble metal nanoparticles using localized surface plasmon resonance (LSPR) spectroscopy, surface-enhanced Raman spectroscopy (SERS), and transmission electron microscopy (TEM) to elucidate structure-function relationships. A recently developed LSPR imaging spectroscopy instrument with an extremely fast camera enables measurement of diffusion constants for individual silver nanoprisms dispersed in water—paving the way for plasmonic particles as dynamic labels in biological systems. Correlated studies involving two or all three of these techniques relate optical properties of the same nanoparticle to its structure. Lastly, singlemolecule surface-enhanced Raman spectroscopy of Rhodamine 6G is explored on lithographically fabricated plasmonic structures.

Understanding the detailed relationship between nanoparticle structure and activity remains a significant challenge for the field of surface-enhanced Raman spectroscopy. To this end, the structural and optical properties of individual plasmonic nanoantennas comprised of Au nanoparticle assemblies that are coated with organic reporter molecules and encapsulated by a SiO(2) shell have been determined using correlated transmission electron microscopy (TEM), dark-field Rayleigh scattering microscopy, surface-enhanced Raman scattering (SERS) microscopy, and finite element method (FEM) calculations. The distribution of SERS enhancement factors (EFs) for a structurally and optically diverse set of nanoantennas is remarkably narrow. For a collection of 30 individual nanoantennas ranging from dimers to heptamers, the EFs vary by less than 2 orders of magnitude. Furthermore, the EFs for the hot-spot-containing nanoparticles are uncorrelated to aggregation state and localized surface plasmon resonance (LSPR) wavelength but are crucially dependent on the size of the interparticle gap. This study demonstrates that the creation of hot spots, where two particles are in subnanometer proximity or have coalesced to form crevices, is paramount to achieving maximum SERS enhancements.

In this work we perform correlated structural and optical studies of single nanoparticles as well as explore the generality of SMSERS. First, wide-field plasmon resonance microscopy is used to simultaneously determine the LSPR spectra of multiple Ag nanoprisms, whose structure is determined using TEM. Next, the structure-property relationships for well-defined and easily-controlled nanoparticle structures (e.g. monomers, dimers, and trimers) are studied using correlated TEM, LSPR, and SERS measurements of individual SERS nanotags. We present the SER spectrum of reporter molecules on a single nanotag comprised of a Au trimer. It was determined that of 40 individual nanotags, just 19 exhibited SERS. The remaining nanoparticles were established by TEM to be monomers. These results demonstrate that SERS signal is observed from individual nanotags containing a junction or hot spot. Lastly, we explore crystal violet, a triphenyl methane dye that was used in the seminal SMSERS investigations, and re-examine single-molecule sensitivity using the isotopologue approach.

The surface-enhanced Raman excitation profiles (REPs) of rhodamine 6G (R6G) on Ag surfaces are studied using a tunable optical parametric oscillator excitation source and versatile detection scheme. These experiments afford the ability to finely tune the excitation wavelength near the molecular resonance of R6G (i.e., approximately 500-575 nm) and perform wavelength-scanned surface-enhanced Raman excitation measurements of a single molecule. The ensemble-averaged surface-enhanced REPs are measured for collections of molecules on Ag island films. The relative contributions of the 0-0 and 0-1 vibronic transitions to the surface-enhanced REPs vary with vibrational frequency. These results highlight the role of excitation energy in determining the resonance Raman intensities for R6G on surface-enhancing nanostructures. Single-molecule measurements are obtained from individual molecules of R6G on Ag colloidal aggregates, where single-molecule junctions are located using the isotope-edited approach. Overall, single-molecule surface-enhanced REPs are narrow in comparison to the ensemble-averaged excitation profiles due to a reduction in inhomogeneous broadening. This work describes the first Raman excitation spectroscopy studies of a single molecule, revealing new information previously obscured by the ensemble.

Single-molecule dark states are often attributed to photoexcited triplets with scant evidence of the participation of paramagnetic molecules. The photodynamics of blinking single molecules of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) in crystals of potassium hydrogen phthalate (KAP) were compared with the lifetimes of DCM triplet states, likewise in KAP, whose zero-field splitting (ZFS) tensors were fully characterized by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy. Luminescent mixed crystals of KAP were grown from solutions containing 10(-4) -10(-9) M DCM, a model optically nonlinear chromophore. The luminescent dye was localized in the {111} crystalline growth sectors. The photoexcited triplets states of DCM in the heavily dyed (10(-4) M) crystals were analyzed by TR-EPR spectroscopy. The photoexcited singlet states of DCM in lightly dyed crystals (10(-9) M) were analyzed by single-molecule microscopy. Large blue shifts in the absorption and emission spectra of DCM in KAP were interpreted as a consequence of protonation at the dimethylamino nitrogen atom, an assignment supported by calculations of the zero-field splitting (ZFS) tensors of molecules in their triplet states. Experimental ZFS tensors with eigenvalues comparable to those of the computed tensors were determined from the angular dependence of the EPR spectra of DCMH(+) triplets within KAP single crystals with respect to the applied magnetic field. Data from individual growth sectors failed to show magnetically equivalent site occupancies, evidence of the kinetic ordering during growth. The intermittent fluorescence of individual chromophores was analyzed. The distributions of on(off) times were characterized by distributed rates fit to power laws. The lifetime of the triplet states was analyzed from the time decay of the EPR signals between 100 and 165 K. The data were well fit with a single time constant for the signal decay, a result wholly inconsistent with the blinking of single molecules with off times commonly of tens of seconds. Triplet decay was extrapolated to approximately 25 micros at room temperature. Therefore, the assumption that single-molecule dark states originate with triplet excited states is not sustainable for single DCM molecules in KAP.

We present single-molecule confocal microscopy studies of orientational distributions for luminophores isolated in potassium hydrogen phthalate (KAP) crystals. The incorporation of dye molecules that bear no size or shape similarity to the host ions is observed, demonstrating that single-molecule studies on mixed crystals need not be restricted to isomorphous host/guest pairs. Violamine R is oriented and overgrown by the fast vicinal slopes of growth hillocks within the symmetry-related {010} growth sectors and DCM deposits in the {11} growth sectors of KAP. Both mixed crystals exhibit modest absorption dichroism relative to basic pyranine-doped K(2)SO(4). The latter was studied to ensure that a range of orientations was sampled in our experiments. Average orientations determined at the single-molecule level were in close agreement to ensemble-averaged measurements for all three systems, and the chromophore orientational distributions were broader than anticipated, indicating that the crystals incorporate guest molecules in a range of orientations outside the measured ensemble average.

Identifying natural, organic dyes and pigments is important for the conservation, preservation, and historical interpretation of works of art. Although previous SERS studies have demonstrated high sensitivity and selectivity for red lake pigments using various pretreatment conditions, corresponding investigations of yellow lake pigments and paints are relatively sparse. Here, surface-enhanced Raman scattering (SERS) spectroscopy is used to identify a variety of yellow organic dyestuffs and lake pigments in oil paint. High-quality SERS spectra of yellow dyestuffs (i.e., turmeric, old fustic, Buckthorn berries) and corresponding paints could be obtained with or without sample pretreatment using microliter quantities of HCl and methanol at room temperature. However, the SERS spectra of yellow lake pigments (i.e., Stil de Grain, Reseda lake) and their corresponding oil paints were only observed upon sample pretreatment. Ultimately, we demonstrate a reliable sample treatment protocol for SERS-based identification of turmeric, old fustic, Buckthorn berries, Stil de Grain, and Reseda lake as well as for microscopic samples of the corresponding oil paints.

This perspective presents recent surface-enhanced Raman spectroscopy (SERS) studies of dyes, with applications to the fields of single-molecule spectroscopy and art conservation. First we describe the development and outlook of single-molecule SERS (SMSERS). Rather than providing an exhaustive review of the literature, SMSERS experiments that we consider essential for its future development are emphasized. Shifting from single-molecule to ensemble-averaged experiments, we describe recent efforts toward SERS analysis of colorants in precious artworks. Our intention is to illustrate through these examples that the forward development of SERS is dependent upon both fundamental (e.g., SMSERS) and applied (e.g., on-the-specimen SERS of historical art objects) investigations and that the future of SERS is very bright indeed.

endo-cis-N-Phenylbicyclo[2.2.2]oct-5-en-2,3-dicarboximide was synthesized by a Diels–Alder cycloaddition of 1,3-cyclohexadiene and N-phenylmaleimide in ethyl acetate. 1,3-Cyclohexadiene and N-phenylmaleimide were selected to illustrate the Alder rule, which reflects a preference for endo products and to overcome the difficulties associated with the traditional combination of 1,3-cyclopentadiene and maleic anhydride. This Diels–Alder reaction can be performed on macro- or miniscale, at room temperature for a week or under reflux for 2.5 hours to produce a 91% yield of a white solid that precipitates from solution and can be analyzed without further purification. Typical student results after 1.5 hours of reflux were 1.05 g (78%), mp 203.4–205.3 °C (lit. 204–206 °C). Results from IR and 1H and 13C NMR were consistent with literature values.

The multistep synthesis of 4-hydroxy-4,4-diphenyl-2-butanone from ethyl acetoacetate illustrates the use of a ketal protecting group. Reaction of ethyl acetoacetate with ethylene glycol with p-TsOH in toluene produced the ketal ester. Reaction of the crude ketal ester with two equivalents of phenyl magnesium bromide followed by an aqueous acid workup generated the tertiary alcohol and simultaneously removed the ketal protecting group to produce the hydroxyketone. Our procedure is a modification of a previously published synthesis whose end product was 4,4-diphenyl-3-buten-2-one, the dehydrated analog. Our modifications involved the exchange of toluene for carcinogenic benzene as the azeotropic solvent in the ketal forming step and the alteration in the Grignard workup from water to one with dilute acid. This shortens the experimental sequence by avoiding the isolation and subsequent deprotection of the hydroxyketal intermediate and avoids the complication of the concurrent dehydration. We engaged the stud...