Spectra Of Pigments Research Articles

Heat and light stability of natural yellow colourants in model beverage systems

ABSTRACT The performance of 10 natural yellow colourants at different concentrations (0.001–0.3% w/v) was investigated in a model beverage system at various pH levels (3.5, 5.5 and 7.5) containing 10% w/v of sucrose, with or without ethanol (15% v/v) to simulate both alcoholic and non-alcoholic lemon-like beverages. Thus, the incidence of pH, ethanol and colourant concentration on spectral properties as well as turbidity were evaluated. The λmax was found to be independent from concentration in the tested range (0.001– 0.3% w/v), whereas the specific absorbance at λmax increased linearly. Moreover, the absorption spectrum and λmax of yellow pigments were found to be independent of pH and ethanol. Gardenia, safflower, and curcumin had the highest colour intensity and the lowest turbidity level. Heat and light stability were studied for these as well. Safflower was found to be the most stable colourant against heat (25, 40, 60, 80°C) and light (550 W/m2, 30°C).

Synthesis and fine spectroscopy tuning of the hyperspectral simulation material based on organic anions intercalated Mg-Al layered double hydroxide

Organic anions have been intercalated into the interlayer of Mg-Al layered double hydroxides (LDHs) via a hydrothermal method to tune the visible and near-infrared reflection spectrum of hybrid pigments based on LDHs. The prepared samples were analyzed by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric-differential scanning calorimetry, visible and near-infrared spectrophotometry, and CIE 1976 L*a*b* color scales. The effects of the intercalated organic anions on the structure, morphology, color, and spectral characteristics of Mg-Al LDH materials were systematically investigated. The results show that the colored anions of acid green (AG) and acid yellow (AY) have been successfully intercalated into the interlayer space of LDHs through the anion displacement process. The visible light reflection spectrum of LDH matrix can be easily tuned by adjusting the intercalated concentration of anions and the relative ratio of AG and AY, which leads to the color of prepared samples changing from the original white into blue, yellow, green, and dark black. Combined with the color adjusting ability of organic anions and the high hydroxyl property of LDH substrate, the typical visible and near-infrared spectral characteristics of natural green plants can be accurately simulated. The correlation coefficient of spectrum shape between the prepared pigment and green plant in the whole range of 400–2500 nm is as high as 0.94. Moreover, the thermal stability of LDH materials is improved by the intercalated organic anions, whose spectral properties can withstand high temperatures of at least 250 °C. The hybrid materials could be used as a functional pigment to prepare green camouflage coatings, showing their potential application in the field of hyperspectral camouflage.

Investigation of Pigments on an Indian Palm Leaf Manuscript (18th – 19th century) by SEM-EDX and other Techniques

AbstractThis paper discusses pigments used in Indian palm leaf manuscripts. Formerly it was believed that pigments for palm leaf illustrations were mostly sourced from plant extracts – a believe that is still widespread in India. This paper reports the identification of pigments of an illustrated palm leaf manuscript (eighteenth – nineteenth century) collected from the east coast of India. As Raman spectra of many pigments gave too high background noise, the identification was mostly accomplished through SEM-EDX and FTIR spectroscopy. The analytical findings indicated that both mineral colours and plant extracts were sourced for Indian palm-leaf manuscripts. Analysis indicated the use of lamp black for black, indigo for blue, vermillion for red, orpiment for yellow, china clay for white and a mixture of orpiment and indigo for green coloration in the illustrations. The data has improved our knowledge of historic pigments used in palm leaf illustrations. As some of the pigments are either photosensitive or darken due to atmospheric pollution, necessary preventive conservation measures need to be adopted.

Разнообразие и адаптивные свойства зрительных пигментов рыб с изменяемой окраской роговицы

The properties of the visual pigments in several fish species that possess a unique physiological mechanism of changing the corneal colouration depending on the intensity of ambient illumination are described in the present paper. The absorbance spectra of the visual pigments and the spectral characteristics of the cornea have been measured in masked greenling Hexagrammos octogrammus and five fish species of the order Tetraodontiformes using microspectrophotometry. In all the species, retinal photoreceptors contain a mixture of the rhodopsin (A1) and porphyropsin (A2) pigments in different proportions, from 0 to 100%. The experiments with double cones in a chloride-free medium have revealed a shift of λmax in some of the pigments to the shortwave region (ionochromic effect). This suggests that pufferfishes have one pigment of the LWS type (λmax = A2_567 nm), while greenling has two LWS-pigments (λmax = A2_625, and A1_560 nm). The shift of the spectral absorbance of double cones towards the longwave region in pufferfish, as compared to that in most other shallow-sea fishes, is probably explained by the presence of a changeable yellow-orange carotenoid corneal filter.

Spectral tuning mediated by helix III in butterfly long wavelength-sensitive visual opsins revealed by heterologous action spectroscopy

Absorption spectra of opsin-based pigments are tuned from the UV to the red regions by interactions of the chromophore with surrounding amino acid residues. Both vertebrates and invertebrates possess long-wavelength-sensitive (LWS) opsins, which underlie color vision involving “red” sensing. The LWS opsins have independently evolved in each lineage, which suggests the existence of diverse mechanisms in spectral tuning. In vertebrate LWS opsins, the mechanisms underlying spectral tuning have been well characterized by spectroscopic analyses with recombinant pigments of wild type (WT) and mutant opsins. However in invertebrate LWS opsins including insect ones, the mechanisms are largely unknown due to the difficulty in obtaining recombinant pigments. Here we have overcome the problem by analyzing heterologous action spectra based on light-dependent changes in the second messenger in opsin-expressing cultured cells. We found that WTs of two LWS opsins of the butterfly, Papilio xuthus, PxRh3 and PxRh1 have the wavelengths of the absorption maxima at around 570 nm and 540 nm, respectively. Analysis of a series of chimeric mutants showed that helix III is crucial to generating a difference of about 15 nm in the wavelength of absorption maxima of these LWS opsins. Further site-directed mutations in helix III revealed that amino acid residues at position 116 and 120 (bovine rhodopsin numbering system) are involved in the spectral tuning of PxRh1 and PxRh3, suggesting a different spectral tuning mechanism from that of primate LWS opsins.

Engineering cation vacancies to improve the luminescence properties of Ca14Al10Zn6O35: Mn4+ phosphors for LED plant lamp

AbstractIn the recent years, Mn4+‐doped phosphors for indoor plant cultivation have received extensive concern owing to the far‐red emission that can match well with the absorption spectra of plant pigments. Whereas, many Mn4+‐doped phosphors still face some challenges such as poor light efficiency and low thermal stability. It is an effective way to resolve these problems via cation vacancies engineering. Herein, the Ca14−xAl10Zn6−yO35: Mn4+ phosphors are successfully synthesized by combustion method. The luminescence intensity of Ca14−xAl10Zn6−yO35: Mn4+ phosphor is enhanced through engineering Ca2+ and Zn2+ vacancies according to the charge compensation mechanism. The optimal content of each Ca2+ and Zn2+ vacancy is equal to be 0.3. Furthermore, the defect formation is accompanied with lattice distortion, which plays a vital role in driving the excited phonon traps to reduce the energy loss by non‐radiation transitions. Therefore, the thermal stability of Ca14−xAl10Zn6−yO35: Mn4+ phosphor is also improved via engineering cation vacancies. In addition, the Ca14−xAl10Zn6−yO35: Mn4+ phosphors can be effectively excited by blue light and it exhibits far‐red emission due to the Mn4+ spin‐forbidden 2E → 4A2 transition. The results suggest that the Ca14−xAl10Zn6−yO35: Mn4+ phosphors can have a tremendous potential in indoor plant cultivation.

Seeing red: towards an improved protocol for the identification of madder- and cochineal-based pigments by fiber optics reflectance spectroscopy (FORS)

Fiber optics reflectance spectroscopy (FORS) is commonly used to non-invasively identify madder- and cochineal-based pigments on works of art, but the significant shifts sometimes observed in the position of their diagnostic absorption features can hinder correct interpretation of the spectra. To better understand these shifts, and improve the ability to confidently identify these pigments, a systematic study was carried out to evaluate the effects of different pigment recipes and laking substrates on reflectance spectra. Sixteen different madder- and cochineal-based pigments were synthesized using historical recipes. Each pigment, painted in four different binding media (gum Arabic, linseed oil, beeswax, and egg yolk), was fully characterized by FTIR and HPLC-DAD-MS prior to FORS measurements. The results of the study showed that, in contrast to the absorption features typically used for identification, features in the first derivative transformation of the FORS spectra provided a more robust means of primary identification. In addition, once it has been identified as cochineal, the absorption features in the spectra of cochineal-based pigments could be correlated to the recipe employed, providing a possible means for inferring the method of manufacture and laking substrate from a non-invasive analysis. The results of this study were used to create a decision tree for the identification of madder and cochineal pigments based solely on FORS.

Carotenoids produced by halophilic bacterial strains on mural paintings and laboratory conditions.

Due to the presence of efflorescences and improper microclimate conditions for conservation, pink-pigmented areas were reported in two historic monuments in Northern and Central part of Romania. The aims of the present study were to find the nature of pink pigments observed on the pictorial layer, original and infilling mortar, to investigate the presence of carotenoids both on mural paintings and in the isolated halophilic bacterial strains and to preliminary characterize and identify the producing strains. Their role in the aesthetical biodeterioration of historic monuments was also pointed out. Obtained Raman spectra of the pink pigments extracted both from the isolated bacterial cultures (molecularly identified as mostly related to Halobacillus hunanensis and Halobacillus naozhouensis) and from the mural painting samples contain diagnostic bands of carotenoids. These results were confirmed by FTIR spectroscopy. The strong Raman signal of bacterial carotenoids detected on mural painting indicated their potential use as biomarker molecules in the evaluation of contamination and state of conservation of mural paintings and lithic monuments. Our results contribute to opening a new direction in cultural heritage restoration to assess the conservation status on the basis of interdisciplinary research, starting with spectroscopic methods (Raman, FTIR) and confirmed by microbiological analysis.

DESIGNING AND CONSTRUCTION OF THE SPECTRAL LIBRARY FOR TYPICAL PIGMENTS USED IN CHINESE PAINTINGS

Abstract. Suffered from the environmental changes and human’s influence, Chinese paintings are facing a worrying situation in documentation, which leads urgent into establishing digital archives to realize the permanent preservation of its surface information. Non-destructive identification of pigment types using hyperspectral techniques is the key to ensure the colour restoration scientifically. At present, one commonly used method for spectral identification is to calculate the similarities between unknown spectra and standard spectra in spectral library. The purpose of this study is to establish a spectral library of typical pigments specifically for the information preservation and pigment types identification on surface of Chinese paintings. The main works of the research are: (1) collecting the standard spectra in laboratory and the pigment spectra on surface of real cultural relics as the data source of the spectral library; (2) adding the expert knowledge of traditional pigments and normative descriptions of spectra measurement as part of the spectral library; and (3) constructing the spectral library website with the Browser/Server structure, and by using MySQL database for storage, SSM framework for background building and JSP page for displaying. The significance of the design and construction of the Typical Pigments of Chinese Paintings (TPCP) spectral library is not only to establish a standard spectral library for pigment types identification, but also a digital archive of pigment information on the surface of real cultural relics, to achieve permanent preservation, and provide spectral data sources and pigment information references for conservation workers and researchers in related fields for further research.

A novel method for evaluating sun visible light protection factor and pigmentation protection factor of sunscreens.

IntroductionThe role of visible light (VL) in the process of skin pigmentation by solar radiation has been demonstrated. Sunscreens with the presence of pigments, particularly iron oxide (IO), have a greater protective effect against VL and are, therefore, highly recommended for prevention and treatment of hyperpigmentation disorders. This study aimed to evaluate 33 sunscreen formulations, and through spectrophotometric measurements, proposes new methods to evaluate the VL protection factor and the pigmentation protection factor.Methods33 sunscreen formulations marketed in Brazil were evaluated, including 17 products containing pigments and 16 products with no pigments. Spectrophotometric measurements were taken to determine solar VL protection factor and the pigmentation protection factor, based on the absorption curve of the product, the pigmentation action spectrum and sun spectrum.ResultsSunscreen with pigments presented a higher solar VL protection factor and pigmentation protection factor compared to products without pigment. The statistical analysis showed a strong correlation between the solar VL protection factor and the integral of the absorption curve in the VL range and the transmittance reduction (%) in the same range. The correlation between the VL protection factor and the pigmentation protection factor was also demonstrated.ConclusionThe VL protection factor and the pigmentation protection factor showed to be adequate metrics to estimate the effectiveness of sunscreens in the prevention of the pigmentary effect of solar VL and the pigmentary effect of the complete spectrum of immediate pigmentation.

Raman spectroscopy of natural and cultured pearls and pearl producing mollusc shells

AbstractThe attractiveness of natural and cultured pearls is related, in part, to their colour. Raman spectroscopy was applied to natural and cultured pearls and the shells from 23 species of pearl producing molluscs, thus covering the majority of the natural and cultured pearls and their host animals found in the market today, in order to better understand their colouration and eventually to be able to identify the host animal. Pigments with a polyenic chain were identified in samples from 18 species. The vast majority were found to be a mixture of partially methylated polyenes, similar to those observed in octocorals. Samples with a similar colour but from different animal species can be sometimes related to a significantly different mixtures of such pigments. Only natural pearls from Pinna nobilis were found to be coloured by carotenoids. Raman spectra of other pigments were also observed on samples from four species, possibly linked with pigments from the porphyrin group.

Enhancing photoluminescence properties of Mn4+‐activated Sr4−xBaxAl14O25 red phosphors for plant cultivation LEDs

AbstractNon–rare earth Mn4+‐activated strontium aluminate phosphor is considered to be a promising material for plant cultivation field owing to their advantage of inexpensively, environment friendly, nontoxic, and appropriate spectral range. In this paper, a Sr4−xBaxAl13.99O25:0.01Mn4+,1.4H3BO3 strontium aluminate phosphor is synthesized by a convenient high‐temperature solid‐state reaction. The photoluminescence spectra located at red region with a peak at 655 nm in the range of 600 to 750 nm that can be excited under the excitation of ultraviolet (~346 nm) or blue light (~450 nm). The shape emission band at 655 nm is attributed to the transition of 2Eg‐4A2g. Furthermore, the emission intensity of Sr4−xBaxAl13.99O25: 0.01Mn4+,1.4H3BO3 phosphor is conducted in detail with the variety of Ba2+ doping concentration and the intensity can be enhanced to 140.9% than the Sr4Al13.99O25:0.01Mn4+,1.4H3BO3 when the value of Ba2+ concentration equal to 0.1 mol. In addition, the X‐ray diffraction spectra, element mapping, composition modifying, optical properties, FT‐IR spectra, diffuse reflectance spectra, thermal stability, and fluorescence lifetime are systematically investigated. According to the electro‐luminescent spectra of as‐packaged LED, indicating that the Sr3.9Ba0.1Al13.99O25:0.01Mn4+,1.4H3BO3 phosphor will become a great candidate for plant cultivation LEDs due to the emission spectra match well the plant pigment spectrum.

Colour in bivalve shells: Using resonance Raman spectroscopy to compare pigments at different phylogenetic levels

AbstractSeveral studies have suggested that shell colour may be phylogenetically distributed within the phylum Mollusca, but this pattern is confounded by our ignorance of the homology of colour and lack of understanding about the identity of most molluscan pigments. We use resonance Raman spectroscopy to address this problem by examining bivalve pigments producing a range of colours and compare spectra from taxa at different phylogenetic levels. The spectra of most shell pigments exhibited a skeletal signature typical of partially methylated polyenes, possibly modified carotenoids, with the strongest peaks occurring between 1,501–1,540 cm−1 and 1,117–1,144 cm−1 due to the C═C (ν1) and C–C (ν2) stretching modes, respectively. Neither pigment class nor mineral structure differentiated Imparidentia and Pteriomorphia. Spectral acquisitions for purple pigments for two species of Asaphis suggest that identical or nearly identical pigments are shared within this genus, and some red pigments from distantly related species have similar spectra. Conversely, two species with brown shells have distinctly different pigments, highlighting the difficulty in determining the homology of colour even within a single class of pigments. Curiously, we were unable to detect any Raman activity for green‐coloured shell or pigment peaks for the yellow area of Codakia paytenorum, suggesting that these colours are due to structural elements or a pigment that is quite different from those observed in other taxa examined to date. Our results are consistent with the idea that classes of pigments are evolutionarily ancient but heritable modifications may be specific to clades.

Extensive stacking of DHI-like monomers as a model of out-of-plane complexity in eumelanin protomolecules: Chemical and structural sensitivity of optical absorption spectra

In this work, we study the optical absorption spectra of extensive stacking forms of four molecules known to be among eumelanin basic building blocks: 5,6-dihydroxyindole (DHI), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), indolequinone (IQ) and quinone-methide (MQ). Stacked monomers can be considered as a minimal model of out-of-plane complexity in eumelanin, as complementary to in-plane oligomerization. The choice of a plane wave density functional theory (DFT) approach allows us to address extensive, i.e. several-layer, stacking of these DHI-like monomers at a relatively low computational cost, by treating stacked systems as periodic along the stacking direction. Absorption spectra of stacked monomers display interesting trends in terms of chemical and structural sensitivity, which can shed light on the role of extensive stacking in the transition from the optical properties of small molecules such as DHI to the typical broadband and monotonic absorption spectrum of the eumelanin pigment.

Thermostability of Photosystem I Trimers and Monomers from the Cyanobacterium Arthrospira platensis

The correlation between changes in the circular dichroism (CD) spectra of antenna pigments and the photooxidation kinetics of the primary electron donor (P700) in trimeric and monomeric complexes of photosystem 1 (PS1) after incubation at elevated temperatures was studied to assess the thermostability of photosystem I (PSI) complexes from the cyanobacterium Arthrospira platensis. It was shown that heating of the monomers and trimers to 60 and to 70°C, respectively, caused a 10% decrease in the amplitude of the CD signals of antenna chlorophyll. Thermal disturbance of the spatial organization of the antenna chlorophyll correlated with a lower concentration of photoactive P700. A decrease in the initial rate of P700 photooxidation was observed already after 40°C for both monomers and trimers. This decrease occurred simultaneously with a decrease in the intensity of the carotenoid CD band of trimers but not monomers, which is probably due to the different carotenoid compositions of trimers and monomers. It is assumed that the slowdown of P700 photooxidation kinetics in PS1 complexes at temperatures up to 50–60°C is associated not with impairment of energy migration to the reaction center (P700) but rather with an initial delay of P700 photooxidation due to the changes in the state of the acceptor part of PS1, in particular, the secondary acceptor phylloquinone.

Expression of tyrosinase genes associated with fruiting body formation and pigmentation in Grifola frondosa

Chemical and physical properties (including ultraviolet spectrum) of a brown pigment isolated from Grifola frondosa fruiting bodies were almost identical to those of fungal melanin. From the full genome sequence of G. frondosa , genes involved in the melanin biosynthesis pathway of this polypore mushroom were identified, including tyrosinases ( tyr1 and tyr2 ). Transcriptional analyses showed dramatic changes only in the expression of tyr2 in the primordial stage. Immuno-electron microscopy using anti-tyrosinase antibodies showed that TYR2 localizes in the cell walls of primordia. Therefore, TYR2 may be closely associated with melanin biosynthesis in this polypore mushroom G. frondosa , and melanin might be produced in the cell wall. • Brown pigment from Grifola frondosa fruiting bodies was identified as melanin. • We identified tyrosinases tyr1 and tyr2 from the full genome sequence of G. frondosa. • Transcription of tyr2 showed dramatic increase in the primordial stage only. • TYR2 localized in the cell wall of wild-type primordial cells.

Cellular localization of tolyporphins, unusual tetrapyrroles, in a microbial photosynthetic community determined using hyperspectral confocal fluorescence microscopy.

The cyanobacterial culture HT-58-2, composed of a filamentous cyanobacterium and accompanying community bacteria, produces chlorophyll a as well as the tetrapyrrole macrocycles known as tolyporphins. Almost all known tolyporphins (A-M except K) contain a dioxobacteriochlorin chromophore and exhibit an absorption spectrum somewhat similar to that of chlorophyll a. Here, hyperspectral confocal fluorescence microscopy was employed to noninvasively probe the locale of tolyporphins within live cells under various growth conditions (media, illumination, culture age). Cultures grown in nitrate-depleted media (BG-110 vs. nitrate-rich, BG-11) are known to increase the production of tolyporphins by orders of magnitude (rivaling that of chlorophyll a) over a period of 30-45 days. Multivariate curve resolution (MCR) was applied to an image set containing images from each condition to obtain pure component spectra of the endogenous pigments. The relative abundances of these components were then calculated for individual pixels in each image in the entire set, and 3D-volume renderings were obtained. At 30 days in media with or without nitrate, the chlorophyll a and phycobilisomes (combined phycocyanin and phycobilin components) co-localize in the filament outer cytoplasmic region. Tolyporphins localize in a distinct peripheral pattern in cells grown in BG-110 versus a diffuse pattern (mimicking the chlorophyll a localization) upon growth in BG-11. In BG-110, distinct puncta of tolyporphins were commonly found at the septa between cells and at the end of filaments. This work quantifies the relative abundance and envelope localization of tolyporphins in single cells, and illustrates the ability to identify novel tetrapyrroles in the presence of chlorophyll a in a photosynthetic microorganism within a non-axenic culture.

Patterns of genomic differentiation between two Lake Victoria cichlid species, Haplochromis pyrrhocephalus and H. sp. \u2018macula\u2019

BackgroundThe molecular basis of the incipient stage of speciation is still poorly understood. Cichlid fish species in Lake Victoria are a prime example of recent speciation events and a suitable system to study the adaptation and reproductive isolation of species.ResultsHere, we report the pattern of genomic differentiation between two Lake Victoria cichlid species collected in sympatry, Haplochromis pyrrhocephalus and H. sp. ‘macula,’ based on the pooled genome sequences of 20 individuals of each species. Despite their ecological differences, population genomics analyses demonstrate that the two species are very close to a single panmictic population due to extensive gene flow. However, we identified 21 highly differentiated short genomic regions with fixed nucleotide differences. At least 15 of these regions contained genes with predicted roles in adaptation and reproductive isolation, such as visual adaptation, circadian clock, developmental processes, adaptation to hypoxia, and sexual selection. The nonsynonymous fixed differences in one of these genes, LWS, were reported as substitutions causing shift in absorption spectra of LWS pigments. Fixed differences were found in the promoter regions of four other differentially expressed genes, indicating that these substitutions may alter gene expression levels.ConclusionsThese diverged short genomic regions may have contributed to the differentiation of two ecologically different species. Moreover, the origins of adaptive variants within the differentiated regions predate the geological formation of Lake Victoria; thus Lake Victoria cichlid species diversified via selection on standing genetic variation.

Gaussian decomposition and component pigment spectral analysis of phytoplankton absorption spectra

The absorption spectrum of phytoplankton is an important bio-optical parameter for ocean color hyperspectral remote sensing; its magnitude and shape can be affected considerably by pigment composition and concentration. We conducted Gaussian decomposition to the absorption spectra of phytoplankton pigment and studied the spectral components of the phytoplankton, in which the package effect was investigated using pigment concentration data and phytoplankton absorption spectra. The decomposition results were compared with the corresponding concentrations of the five main pigment groups (chlorophylls a, b, and c, photo-synthetic carotenoids (PSC), and photo-protective carotenoids (PPC)). The results indicate that the majority of residual errors in the Gaussian decomposition are <0.001 m−1, and R2 of the power regression between characteristic bands and HPLC pigment concentrations (except for chlorophyll b) was 0.65 or greater for surface water samples at autumn cruise. In addition, we determined a strong predictive capability for chlorophylls a, c, PPC, and PSC. We also tested the estimation of pigment concentrations from the empirical specific absorption coefficient of pigment composition. The empirical decomposition showed that the Ficek model was the closest to the original spectra with the smallest residual errors. The pigment decomposition results and HPLC measurements of pigment concentration are in a high consistency as the scatter plots are distributed largely near the 1:1 line in spite of prominent seasonal variations. The Woźniak model showed a better fit than the Ficek model for Chl a, and the median relative error was small. The pigment component information estimated from the phytoplankton absorption spectra can help better remote sensing of hyperspectral ocean color that related to the changes in phytoplankton communities and varieties.

Using Raman spectroscopy to estimate the dates of carbon-based inks from Ancient Egypt

In the first study of its kind, we previously investigated Raman spectra of 17 papyri from the Columbia University Libraries collection, written in carbon-based black ink with well-established dates between 400 BCE and 1000 CE. Using a simple two-peak spectral model for the Raman spectrum of carbonaceous materials, we discovered small but statistically significant systematic variations in the spectral parameters with manuscript date. The purposes of this report are to present: (1) more-powerful statistical examinations of the correlation based on the two-peak spectral model and on a new three-peak model which includes spectral effects for amorphous carbon; (2) new data for additional ancient manuscripts, broadening the investigation; (3) a method for potentially exploiting the observed correlation to predict the dates of undated manuscripts of similar character; and (4) a hypothesis as to the underlying chemical basis of the observed spectral changes. We apply both linear and multiple linear regression analyses to the spectral models. We show that the three-peak model fits the experimental data much better than the two-peak model. Through the use of multiple linear regression, the three-peak model reduces significantly the overall correlation error. We find that our ability to predict dates for manuscripts that are similar to those of the study over the date range 400 BCE to 1000 CE is limited by intrinsic variations in the character of the pigments to about ± 118 years with 68% statistical confidence (or ± 236 years with 95% confidence). We confirm the observed correlation and prediction capabilities through a blind study of six additional ancient manuscripts and two late 19th- or early 20th-century fakes in the Columbia University collection. The data suggest that chemical degradation of amorphous carbon and chemical modification of graphitic crystals may both contribute to the observed spectral differences. This would also account for the magnitude of the variance in spectral character we observe, which possibly reflects differences in pigment manufacture processes as well as the effects of differences in chemical micro-environments after writing. The chemical degradation hypothesis receives additional support from previously unreported examination of two manuscripts from the collection of the Brooklyn Museum. The older of these, dated through conventional papyrological methods to 2323-2150 BCE, deviates from the linear regression models, but in a way consistent with chemical (i.e., non-linear) degradation. For comparison, we report Raman scattering for 8 different modern inks using the same protocol we developed for the ancient inks. Using statistical techniques based on Gaussian Mixture Modeling, we demonstrate differences between the spectral character of the inks of our base study and those prepared from modern reference pigments. This allows us to define a mathematical quantity that can distinguish between spectra of pigments that are spectrally similar to the ancient pigments studied here and those that are not. This new and non-destructive method produces a statistically robust indication of the antiquity and rough period (e.g., Ptolemaic, Roman, etc.) of the inks in question, if not the precise date. We hypothesize that the potential for greater precision is likely limited by the intrinsic variations in the spectral characteristics of the inks from manuscript to manuscript.