review article microanalysis of organic pigments in

Review ArticleMicroanalysis of Organic Pigments in Ancient Textiles bySurface-Enhanced Raman Scattering on Agar Gel Matrices

Marilena Ricci1 Cristiana Lofrumento1 Emilio Castellucci12 and Maurizio Becucci12

1Department of Chemistry ldquoUgo Schiffrdquo University of Florence Via della Lastruccia 3-13 50019 Sesto Fiorentino Italy2European Laboratory for Non-Linear Spectroscopy (LENS) Via N Carrara 1 50019 Sesto Fiorentino Italy

Correspondence should be addressed to Maurizio Becucci mauriziobecucciunifiit

Received 31 January 2016 Revised 30 March 2016 Accepted 3 April 2016

Academic Editor Christoph Krafft

Copyright copy 2016 Marilena Ricci et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We review some new methods based on surface-enhanced Raman scattering (SERS) for the nondestructiveminimally invasiveidentification of organic colorants in objects whose value or function precludes sampling such as historic and archeologicaltextiles paintings and drawing We discuss in detail the methodology we developed for the selective extraction and identificationof anthraquinones and indigoids in the typical concentration used in textiles by means of an ecocompatible homogeneousnanostructured agar matrix The extraction system was modulated according to the chemical properties of the target analyte bychoosing appropriate reagents for the extraction and optimizing the extraction time The system has been found to be extremelystable easy to use and produce easy to store and at the same time able to be analyzed even after long time intervals maintaining itsenhancement properties unaltered without the detriment of the extracted compoundHighly structured SERS band intensities havebeen obtained from the extracted dyes adopting laser light excitations at 5145 and 785 nm of a micro-Raman setup This analyticalmethod has been found to be extremely safe for the analyzed substrates thus being a promising procedure for the selective analysisand detection of molecules at low concentration in the field of artworks conservation

1 Introduction

Identification of dyes used in works of art belonging todifferent molecular classes is essential for dating restor-ing and conserving artwork and for studying art historyin general The importance of the cultural artifacts suchas archeological and ancient textiles drawings and paint-ings requires techniques that can lead to the unambiguousidentification of natural dyes from microscopic samples ordirectly from artworks The analytical technique that iscurrently most common for the identification of historicaldyes is high-performance liquid chromatography (HPLC) [1ndash3] Although HPLC is a highly specific and very sensitiveseparation technique laborious extraction and separationsteps could induce molecular changes with a risk of sampleloss in separation steps In addition its sample requirements(approximately 5mm of a thread for textile samples) make itless than ideal for those cases where no or little sampling isallowed Noninvasive and spectroscopic methods of analysis

are usually limited to UV-visible absorption or fluorescencespectroscopy and carried out by means of fiber optics probesand they yield spectra that typically exhibit a poor specificityInstead vibrational spectroscopies can provide very specificinformation on the different materials However infraredabsorption (or reflectance) is a vibrational spectroscopictechnique not ideal for such type of applications becauseof matrix interference effects Raman spectroscopy (RS) isanother vibrational spectroscopy method It is based on theobservation of weak side-bands in the radiation scatteredby the sample upon excitation with monochromatic lightRaman spectroscopy requires no sample preparation andoffers the potential of noninvasive analysis that is a clearadvantage when no sampling is allowed However only alimited number of Raman spectra of natural dyes are availablein the literature [4ndash7] most of the published body of workdeals with inorganic pigments [8ndash11] The two major factorsthat have hampered the widespread application of Ramanspectroscopy for the identification of natural organic dyes

Hindawi Publishing CorporationJournal of SpectroscopyVolume 2016 Article ID 1380105 10 pageshttpdxdoiorg10115520161380105

2 Journal of Spectroscopy

are their very high tinting power (ie they are likely to befound in works of art in extremely low concentrations) andthe strong fluorescence signal generated by excitation withvisible light even when red or near red emitting laser is usedand that often obscures the inherently weakRaman scatteringsignal

A phenomenon that can significantly enhance the Ramansignal intensity is the surface-enhanced Raman scattering(SERS) effect The Raman scattering efficiency of moleculecan be enhanced when the molecule is effectively interactingwith a nanostructured metal surface and it is spatially con-fined within the electromagnetic field of the localized surfaceplasmon resonance (LSPR) of the nanostructured system[12 13] The localized plasma resonance furnishes a mean tofunnel electromagnetic energy in the proximity of the surfaceof the metallic nanoparticles (NPs) such that light scatteringphenomena of species adsorbed on the metallic surfacecan benefit from a substantial increase in the incomingand outgoing (diffused) electromagnetic fields SERS effectcan give enhancements of Raman signals greater than 8orders of magnitude Also SERS is a selective spectroscopictechnique as only molecules effectively interacting with theNPs show enhanced signals In our specific application ondyed textile fibers the SERS effect is observed only for thedye molecules and not for the substrate Finally it mustbe mentioned that the presence of metal NPs provides avery efficient way to quench fluorescence from the excitedmolecules thus removing a further serious limitation for theapplication of Raman spectroscopy to dye molecules [12 13]Therefore SERS is also very effective for discrimination ofmolecules of different nature present in artistic samples andthe enhancement of the signal enables us to detect even tracequantities of materials

Raman experiments can be carried out also at excitationfrequencies that are close to (or even resonant) the electronicexcitation frequency of the molecule under investigationIn that case we are dealing with the so-called ldquoresonanceRamanrdquo effect that produces much larger signals for spe-cific vibrational modes [14] Surface-enhanced [resonance]Raman scattering (SE[R]RS) is known to produce furtherenhancement of the Raman signal When the LSPR of theenhancing substrate is also in the proper energy region theSERRS enhancement factor is roughly the product of theenhancement factor for nonresonant SERS and the resonanceRaman spectrum intensification factor of the molecule

In order to improve the quality of the enhancementof the Raman scattering and the reproducibility of thespectra a wide class of nanostructured substrates such asrough electrodes [15] colloids [16] nanoisland films [17 18]nanostars [19 20] nanorods [21] and other nanocompositetechnological supports have been carried outThese advance-ments together with solid phase microextraction techniquesthat use synthetic and natural polymeric materials [22] haveallowed researchers in the field of cultural heritage to achieveconclusive identification of dyes from artworks with greatmolecular selectivity and specificity

The first applications SERS methods for dyes identifica-tion on textile fibers were still not enough sensitive to allownondestructive operations [23] A first significant step for

the increase of sensitivity of this method was represented bythe work of Leona [24] Very small (tens of micrometers)samples containing mordant dyes were treated with HFvapors in order to detach the dye from the mordant Then adroplet of colloidal solution containing silver NPs was addedto the sample and the dye was free to move and to bindthe NPs Other methods were developed that use a densedispersion of silver NPs directly on the fibers leading to anirreversible contamination of the original sample [25 26]An innovative and effective method for dyes microextractionfollowed by SERS dyes identification was recently presented[27] It was based on the use of common hydrogels basedon hydroxymethacrylates (contact lens blanks) loaded withwater organic solvents and chelating agents as extractionmedia Another method was presented based on the appli-cation to the sample surface of a cellulose film loaded withsilver NPs [28] The film dries in minutes after applicationand SERS spectra of anthraquinonic dyes present on thesurface were obtained with good sensitivityThe dimensionalchanges associated with the drying process possibly inducesmechanical stress on the sample surface leading to thedetachment of small fragments The film self-detaches or ispeeled off by the operator its formulation was optimizedin order to minimize the gel penetration in the samplesto obtain possibly its complete detachment and to limitthe detachment of small particles from the sample surface[29] Comprehensive reviews on the application of SERSto forensic science and cultural heritage have been alreadypublished [30 31]

This review will discuss the nondestructive microex-traction procedure we developed for dye extraction fromtextile fibers based on the use of an environmentally friendlynanocomposite hydrogel to provide a new tool for thoseresearchers who would like to embark on this kind ofwork using the SERS technique We will present the resultsobtained on mordant dyes (anthraquinones) and vat dyes(indigoids) used both in mockups prepared in the laboratoryand in ancient textiles

2 Materials and Methods

21 Dyeing of Textiles Dyes can be divided into three differ-ent categories (mordant vat and direct dyes) depending onthe procedure according to which they are applied to fabricsIn what follows we are providing information of the differentclasses of natural organic colorants used in textile dyeing inorder to clearly outline the approach we are using

Colorants named ldquomordant dyesrdquo do not have a strongchemical affinity for the textile fibers which therefore requirebeing treated prior to the dyeing stage involving a two-stepchemical reaction A solution of a ldquomordantrdquo is first usedto impregnate the fibers allowing the metal ion to becomecomplexed to appropriate functional groups in the structureof the textile During the dyeing process the colorant inter-acts with themordant-fiber complex via ionic and coordinatecovalent bonds to form insoluble brightly colored speciesThe complex thus formed within the fibers does not easilywash out of the textile and the resulting dyeing is thereforerelatively fast The mordant helped the dye bite onto the

Journal of Spectroscopy 3

fiber so that it would hold fast during washing A schemeof the binding mechanism for alizarin on cotton fibers isshown in Figure 1 Aluminum iron tin chromium or copperions as well as tannins are examples of mordants one ofthe substances most commonly employed in ancient timesfor this purpose was potassium alum (KAl(SO

4)2sdot12H2O)

but iron sulfate (FeSO4sdot7H2O) and tin chloride (SnCl

2) were

often used as well Commonly employed in association withmordants are dye-assistants such as cream of tartar or oxalicacid which brighten the colors protect the fibers and helpthe absorption of mordants Mordant dyes can be used withwool silk and protein yarns while cellulose fibers such aslinen and cotton have to be chemically modified beforedyeing The vast majority of natural colorants belongs tothis category and yields different colors when combined withdifferent mordants a typical example is represented by themadder lake which can produce red orange or violet shadeswhen associated with aluminum tin and iron mordantsrespectively

Colorants belonging to the ldquovat dyesrdquo class are water-insoluble but under reducing conditions they can be con-verted into a leuco form soluble in alkali Nowadays thereducing reagent used is sodiumdithioniteNa

2S2O4 Immer-

sion of the textile into the dye solution allows the dissolvedmolecules of colorant to penetrate the fibers After that uponremoval of the wet dyeing from the bath and exposure toatmospheric oxygen these substances can be oxidized backto their colored forms which thanks to their insolubility inwater are trapped on the surface of the fiberThe well-knownindigo and woad blue colorants as well as Tyrian purplebelong to this category The relevant chemical reactionsinvolved in the dyeing process with indigo are outlined inFigure 2

Even if direct dyes are applied directly to the fiber withoutany special treatment usually they are less wash- and lightfastthan vat or mordant dyes Examples of direct dyes includeturmeric and saffron which can be fixed to all fibrousmaterials in aqueous solution

211 Mockups Dyed with Anthraquinones Three pieces ofcotton fabric were prepared according to traditional dyeingmethodologies by using alizarin (Sigma-Aldrich) purpurin(Sigma-Aldrich) and carminic acid (Sigma-Aldrich) Thetextiles have been treated prior to the dyeing step with analum (Zecchi Firenze) mordant solution in distilled water (JT Baker HPLC Gradient Grade) After the dyeing step theyhave been thoroughly washed with distilled water a couple oftimes and allowed to dry

212 Mockups Dyed with Indigoids Pieces of cotton havebeen dyed according to the recipe reported by Schweppe[32] The dyeing liquor was prepared by stirring 15 g ofindigo powder with 75mL of warm water in a beaker glassuntil it formed a paste In a second vessel 30 g of sodiumhydroxide NaOH was dissolved in 120mL of warm waterA portion of this solution (60ndash70mL) was poured overthe indigo paste stirring vigorously Then 30 g of sodiumdithionite Na

2S2O4 was added while keeping stirring and

1 L of warm water was added while stirring carefully Thismixture was heated to 55∘C Clean pieces of cotton wereimmersed in warmwater until the fabric was thoroughly wetThe fabric was then put inside the dyeing liquor and keptin the dye-bath for some minutes The pieces of cotton werethen taken out of the vat squeezing the liquor out thoroughlyWhen the fabric comes out of the vat it has a typical green-yellow color which turns blue when exposed to air A specialprecaution was used with the Tyrian purple indigoid dyeThe dyeing procedure adopted was similar to the previousone for indigo using a colorantNaOHNa

2S2O4ratio equal

to 1 2 4 Since Tyrian purple is a mixture of brominatedindigoids 6-bromoindigo (MBI) and 661015840 dibromoindigo(DBI) it is subject to debromination because of the actionof UV-light For this reason when the reagents were heatedto 55∘C the light was turned off and the flask was wrappedin aluminum foil A yellow-greenish homogeneous solutionwas obtained and the wet fabric was put into the dyeingbath for 15 minutes Then it was removed from the flask andexposed to air while still protected from light After one hourof air exposure the fabric was rinsed in aqueous solution andallowed to dry

22 Silver Nanoparticles for SERS We used the standardchemical synthesis of Ag nanoparticles (AgNPs) devised byLee and Meisel [16] In order to improve the productionof suitable nanoparticles (NPs) with an average diameter of40 nm we took care of placing the reaction flask after thereduction step in an ice bath [33] The average diameterof the particles was checked from their UV-Vis absorptionspectrum which showed a 100 nm broad peak centered at418 nm in good agreement with previous reports [16]

23 Agar Gel The agar gel usage as the extraction mediumwas suggested by its intrinsic safety for the operator and itswidespread use for the cleaning of artistic materials [34] Itwas prepared from agar-agar a commonly gelling materialused for food preparation Agar-agar is a polymer containingmostly D-galactose units Agar-agar was repeatedly washedin order to remove possible contaminants and chlorideions The gel can be easily prepared by mixing 1 g of agar-agar with 50mL of water warming it for few seconds ina common microwave oven (300W power) and coolingdown the viscous solution in a convenient glassware (Petridish) The agar gel becomes rapidly rigid and stable at roomtemperature It is important to notice that the agar gel isa very good substrate for bacteria cultures so its practicallifetime could be very short and SERS experimental resultscan be altered by the presence of such kind of contaminationA convenient practice resulted to be using the colloidalsolution containing AgNPs in place of water for the agargel preparation It provides a double advantage On one sidethe AgNPs act as a bacteriostatic agent thus preventing theagar gel biological contamination we have found that thiskind of material (Ag-agar) stored in closed containers canbe maintained at room temperatures in the dark for a longtime (a few months) On the other side the agar gel used asdyes extraction medium already carries the AgNPs needed


HO OH

OH

Textile fibers (cotton)

Mordant (alum)

Alizarin

Al3+

O

O

Ominus

Figure 1 Chemical structure of alizarin anion and the bonding mechanism to the cotton fiber mediated by the presence of the Al3+ ion fromthe mordant

H

H

H

H

N

N

N

N

O

O

OH

HO

O2

Na2S2O4NaOH

Leuco-indigo water soluble unstable in air

Diketonic form no solubility in water stable

Figure 2 Chemical reactions involving indigo during the dyeing process The indigo blue dye is the diketonic form

for the SERS measurements Anyway droplets of the AgNPscolloidal solution can be poured in the top of the agar gelbeads we use after the extraction process in order to furtherimprove SERS measurements

24 Sample Extraction The agar gel can act as a suitableextraction substrate for dyes on textile fibers Clearly wateralready present in the agar gel is not per se a suitable solventfor the dyes extraction Different extraction strategies canbe used for different dyes classes Here we are reporting

examples of studies on anthraquinonic [35 36] and indigoiddyes [37]

Anthraquinonic dyes like alizarin and carminic acid arebound to textile fibers by the use of a mordant like alum thatintroduces Al3+ ions These ions act as a bridge between thefiber and the dye molecule (Figure 1) Both molecules can betentatively transferred from the textile fiber to the agar geleither by using a solvent in which they are highly soluble (egan alcohol) or by detaching them from the fiber by specificchemical processesTherefore in the first approach we addeda few ethyl alcohol droplets to the Ag-agar and then we place


it in contact with the fiber for a suitable amount of time (theextraction time was 30 minutes) In the second approachwe tried to detach only the dye from the fiber Even thoughthe chemical etching with HF method was already wellestablished [23] we did try to avoid it as it requires the useof highly toxic chemicals in sealed chamber and the removalof fibers from the original artifact We used a polydentatechelating agent ethylenediaminetetraacetic (EDTA) acid asthe reactant is able to detach the dye by selective removalof the Al3+ ions from the system We added 05 g of EDTA(or equivalent amount of its disodium salt) to the mixturedescribed above during the agar gel preparation The use ofEDTA leads to an acidic system (pH asymp 3) while the use of itsdisodium salt leads to solutions at pHvalues almost neutral Abead (a small cube of 5mm side) of Ag-agar containing alsoEDTA was then placed in contact with the textile for sometime (the extraction time ranges from 15 to 30ndash50 minutesdepending on the textiles being analyzed)

Indigo and related dyes (like Tyrian purple) are water-insoluble dyes that are dissolved in the textile fibers in theirreduced leuco form Once dispersed within the fiber thenatural oxidation process due to the exposure of atmosphericoxygen reverts the dye to its colored form which then sticksto the fiber We added to the Ag-agar gel the chemicalreactants used for the chemical reduction of the indigoiddye Therefore during the contact of the bead we tried toconvert indigo into its leuco form which is water soluble thuspossibly transferable to the agar gel Specifically we addedto the bead a droplet of the NaOHNa

2S2O41 2 ww water

solution described above The bead was then applied to thetextile fibers covered with a glass to reduce evaporation andlet in contact for 5ndash15 minutes

In both cases after the chosen contact time with the fiberthe bead (still wet) was removed from the textile and let dryin air In about 301015840 the bead loses most of its water content(we measure a loss of about 92 in weight) and reducesconsiderably its dimensions (from a 5mm size cube it goesdown to a 1 or 2mm side film about 02mm thick) Thedried bead was then moved to the Ramanmicroscope for themeasurement of the SERS spectrum

25 SERSMeasurements Weused amicro-Raman spectrom-eter RM2000 from Renishaw operating with laser excitationeither at 514 or 785 nm and equipped with a CCD detec-tor thermoelectrically cooled It operates in backscatteringgeometry and uses a 50x microscope objective from LeicaThe typical dimension of the active area of the sample is about2 120583m and the used laser power is in the order of 20ndash200 120583Wat the sample

3 Results and Discussion

The Ag-agar gel shows an open and porous structure withchannels of 50ndash200 nm diameter The AgNPs can be foundin the support either as isolated particles or as aggregatestheir sizes range between 50 and 450 nm in accordance withthe UV-Vis absorption spectrum A high-resolution imageof this nanostructured material (Figure 3) was obtained by

Figure 3Helium ionmicroscope image of the native agar gel loadedwith Ag nanoparticles

using a Helium ion scattering apparatus (Orion Plus by CarlZeiss) a system well suitable for the characterization of softmaterials without any preliminary metallization treatmentThe experimental details for sample preparation and themeasurement settings are described in our previous report[37]

Upon a convenient contact time with the fibers theAg-agar bead was removed from the sample and allowedto dry in air thus decreasing in volume It is a factorthat positively affects the Raman signal as it correspondsto an effective sample concentration process and also bydecreasing the distance between the metal NPs it favors thepresence of stronger local electric fields [13] Also if thedehydration process is not completed the dimension of thebead is not constant and the focusing on the sample in themicroscope is troublesome As a final remark all our testsdemonstrated that the agar gel can be conveniently used asSERS substrate for microextraction as it shows an intrinsicnegligible RamanSERS signal that makes the measurementspractically background-free

The first tests for our microextractionconcentrationSERS determination approach were carried out on the mock-ups prepared with the alizarin dye [35] These tests werefurther extended to include different kinds of fibers andsubstrates [36] The preliminary tests were mostly orientedto verify the extraction efficiency and the safety with respectto the textile fiber We found that both the use of ethanoland EDTA solution as an extraction-promoting reagent onthe Ag-agar beads made it possible to observe the SERSsignal from alizarin already after 10-minute extraction timeIn difficult cases the extraction time can be as long as 50minutes In both cases during extraction the bead must becovered in order to prevent fast evaporation of the solvents

We report in Figure 4 the SERS spectra obtained fromthe dried beads after extraction of the red dye from apre-Columbian textile sample with Ag-agar gel spiked witheither ethanol or disodium-EDTA solution Both spectracorrespond to the well-known Raman spectrum of the


Ethanol extraction

200 600 1000 1400 1800

Raman shift (cmminus1)

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(a)

EDTA assisted extraction


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(b)

Reference


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(c) (d)

Figure 4 SERS spectra (514 nm excitation) obtained from the dried beads after extraction of the red dye from a pre-Columbian textile samplewith Ag-agar gel spiked either with ethanol or disodium-EDTA solution ((a) and (b) resp) or from reference alizarin solutions in ethanol(c) (d) The textile sample is shown with a 25mm ruler on top the 5mm dimension agar gel bead is placed close to the ruler

monoanionic species of the alizarin dye [38] However someattention is neededwhen the experimental data are evaluatedAlizarin is an acid and it is well known that different speciesexist in solution at different pH values Alizarin exists as aneutralmolecule in acidic solutionswhile in neutral solutionsthe most abundant species is the monoanionic one while atpH above 10 the dianionic species is dominant Alizarin isalso an acidochromic molecule that changes its color withpH the neutral molecule is red the monoanion is yellowand the dianion is blue Thus while performing Ramanexperiments on alizarin two key factors must be taken intoaccount [38] The first is the pH as it controls the effectivespecies present in solution the second is the excitationwavelength used for the Raman experiment The chemicalnature of the dye changes with pH then both its color and thepossible resonance Raman effects change as well All of thesephenomena will reflect directly on the shape and intensityof the different vibrational bands observed in the Ramanspectrum Therefore a very good knowledge of the chemical

system is needed and a clear feeling of the relevance of thedifferent experimental parameters is also mandatory In thiscontext the prediction of Raman spectra of alizarin and otherdyes by density functional theory has provided a systematicinformation on these systems and assisted the interpretationof the experimental data collected by SERS [38 39]

Additional tests were carried out concerning the alizarinextraction from textile substrates made with different fiberswood and silk We added the EDTA reagent to the Ag-agar bead for both the considered textile substrates andwe left the agar beads in contact with the fibers for 50minand 15min respectively We succeeded in acquiring excellentSERS spectra of alizarin in both cases thus confirming thecapability of the procedure to extract alizarin from differenttextile fibers [36]

The safety of the process with respect to the textile fiberwas verified by the determination of the color of the fiberin the sampling area before and after the contact with theAg-agar bead Colorimetric measurements on the textile


200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)

Figure 5 SERS spectrum (514 nm excitation) of Tyrian purple extracted from a cotton fiber mockup by using an agar gel bead containingthe leuco-reduction chemicals (a) and Raman spectrum indigo crystals (b)

fiber were made by using a CM-2600d Konica-Minoltaportable spectrophotometer equipped with the integrativesphere and aXenon lampThemeasurement aperture is 3mmdiameter the light that reaches the detector is reflected by theilluminated surface with an angle of 8∘ Color coordinates arebased onCIE 119871lowast119886lowast119887lowast system using an illuminantD65with anobserver angle of 10∘ The value of Δ119864lowast = 23 corresponds toa just noticeable difference in color [40] The results reportedin Table 1 demonstrate that the fibers are virtually nonalteredby our extraction process

After extraction traces of the chemical components of thebead were searched on the textile surface by spectroscopicmethods (ATR-FTIR andX-ray fluorescencemethods) X-rayfluorescence analysis was performed with a Bruker TRACeRIII-SD spectrometer and the spectra were acquired in 50seconds at 40KV15120583A on the rhodium X-ray tube Noevidence for the presence of silver on the fiber was detectedATR-FTIR did not reveal any surface contamination from theagar gel

The first test for extraction of indigoids by water solu-bilization of the dye in its reduced leuco form was carriedout on a mockup textile purposely prepared of Tyrian purple[37] The test is important in order to assess the extractionefficiency and the invasiveness of the process with respectto the textile sample Even though the chemical reagentsinvolved are quite strong (water solution of NaOH andNa2S2O4) we consider them rather safe for the textile fiber as

they are used also in the original dyeing process Also theyare applied only in very small amounts and in a limited areaFinally in the case of relevant contamination being watersoluble they can be washed out easily

The efficiency of the extraction process was measuredby looking at the SERS spectrum that was possibly obtainedfrom the dye extracted on the bead The SERS spectrumobtained from the dye extracted on the bead and measuredon the dried bead with the micro-Raman spectrometer upon514 nm excitation is reported in Figure 5 The excitationconditions we used do not allow for the best sensitivity in theindigo detection by Ramanmeasurements as it is well known

that electronic resonance effects strongly enhance the indigoRaman signal when a red excitation is used [41] However avery good signal to noise ratio is obtained in the observedspectrum It is quite relevant to appreciate that the extractionprocess does not lead to the decomposition of the Tyrianpurple (661015840dibromoindigotin) dye It is well known that thisdye can decompose by the breaking of the C-Br bonds Thebands corresponding to theC-Br stretchingmode at 305 cmminus1are clearly observed in the SERS spectrum

The method was then applied to a small piece of bluewire in wool silk silver and gilded silver obtained fromthe Medicirsquos XVIth century tapestry (Joseph escaping fromPotipharrsquos wife designed by Bronzino) The Ag-agar beadcontaining the leuco-reagents was applied for 10 minutes tothe fiber No sensible discoloration was appreciated on thefiber after the bead removal The SERS spectrum obtainedfrom the dried bead is reported in Figure 6 and it clearlyshows the characteristic pattern of bands associated withindigo (shown in Figure 5(b))

The same tests for the safety of the extraction proceduredescribed above were repeated also in the case of thisseries of measurements As reported in Table 2 the changesof chromatic properties of the mockup textile fiber uponextraction are negligible even for extraction times muchlonger than those needed for obtaining a sample for SERSmeasurements (10 minutes)

X-ray fluorescence analysis revealed a single detectablesignal upon application of the extraction bead It was relatedto Sulphur and it was observed only after 301015840 contact timeATR-FTIR did not show any evidence of surface contamina-tion

Finally we verified if the methods we propose werespecific for the different dyes It is quite clear that extractionpromoted by the presence of a solvent for example ethanolis not specific All the dyes that are not water soluble but havesome affinity with organic solvents can be extracted even ifwith different efficiency We did try to use the EDTAmethoddeveloped formordant dyewith themockupdyedwith indigoand no SERS signal from indigowas detectableThe use of the


200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)

Figure 6 Upper trace Raman spectrum from the blue thread of an XVIth century tapestry (Italian artwork) shown in (b) Lower trace SERSspectrum (514 nm excitation) of indigo extracted from the same sample by using an agar gel bead containing the leuco-reduction chemicalsAdapted from [37]

Table 1 Change in color (CIE 119871lowast119886lowast119887lowast system) of the cotton mockup dyed with alizarin upon extraction with the Ag-agar gel added witheither ethanol or disodium-EDTA solution

Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast

Ethanol15 minutes 4439 4573 2102 006 minus004 minus003 008

Disodium-EDTA30 minutes 4508 459 208 008 minus003 minus004 009

Table 2 Change in color (CIE 119871lowast119886lowast119887lowast system) of the cotton mockup dyed with indigo upon extraction with the Ag-agar gel added with theleuco-reagents

Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast

Point 1601015840 contact time 3636 minus159 minus2251 000 011 minus042 043

Point 21201015840 contact time 3729 minus177 minus2230 minus016 minus003 018 024

leuco-reagents on the alizarin mockup have made the fiberto change temporarily color from red to blue (the high pHvalue of the leuco-reagents solution induced deprotonation ofalizarin) but no extraction of the dye was observed

4 Conclusions

The paper summarizes the development of a SERS substrate(synthesized by mixing a hydrogel agar-agar with a colloidal

dispersion of silver nanoparticles) used to extract minimalamounts of dye molecules from textiles The intent is toprovide a simple efficient and safe method to extractcolorants from textiles fibers of historical artistic andarcheological interest exploiting then the high sensitivity ofSERS spectroscopy for their identification The method wasfound to be very efficient and has advantages with respect toother possible methodologies [23ndash29] It is a nondestructivemethod that is possibly applied directly on the original


fibers without sampling The safety of the methodology wasassessed by means of X-ray fluorescence ATR-FTIR andcolorimetric analyses that confirmed that no residuals ordiscoloring effects have been observed on the textile surfaceafter the microextraction once the extraction process hasbeen optimized with tests on mockups The simplicity of theprocedure to perform SERS measurements the stability ofthe dry nanocomposite gel and the strong enhancement ofthe observed SERS signal are factors that assure the detectionand the recognition of dyes The high level of enhancementachieved is due also to the shrinkage of Ag-agar gel structureupon drying which concentrates the extractedmolecules andpossibly favors the interaction of silver nanoparticles thatcreate high plasmon density sites Moreover using a gel as amedium for the solvent mixture allows for a more localizedand controlled microextraction essay which involves theanalysis of extremely small areas of the object under study

Furthermore the possibility of selecting different solventsand componentswithin the gel structure for the improvementof the extractive performance of the nanocomposite matrixoffers the possibility of tailoring the gel for the extraction ofspecific molecules and for the potential discrimination andrecognition of single components of a mixture

In particular the efficacy of the proposed methodologyhas been widely demonstrated by its application to severaltextiles of artistic relevance validating the suitability of theproposed method on the investigated samples The highefficiency of this methodology confirms that the tailored gelextraction is a promising nondestructive technique for SERSidentification of dyes

Further studies are under way considering other sub-strates for extraction in order to extend the application of themethod to other classes of materials of artistic and historicalinterest such as illuminated manuscripts paintings andsculptures As a possible development of this method weforesee its integration with current cleaning methods basedon the use of agar gel This would allow for simultaneouscleaning and sampling actions to be followed by instrumentalcharacterization of the removed materials

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was supported by Italian MIUR (PRIN Grant2010329WPF 007) and by Ente Cassa di Risparmio di Firenze(Grant no 20140405A22028044) Opificio delle Pietre Dure(Florence I) and the Museo del Tessuto (Prato I) madeavailable samples of ancient textile artworks for our researchTheir support is kindly acknowledged

References

[1] I Degano E Ribechini F Modugno and M P ColombinildquoAnalytical methods for the characterization of organic dyes

in artworks and in historical textilesrdquo Applied SpectroscopyReviews vol 44 no 5 pp 363ndash410 2009

[2] M R van Bommel I V Berghe AMWallert R Boitelle and JWouters ldquoHigh-performance liquid chromatography and non-destructive three-dimensional fluorescence analysis of earlysynthetic dyesrdquo Journal of Chromatography A vol 1157 no 1-2pp 260ndash272 2007

[3] Z C Koren ldquoHPLC analysis of the natural scale insect madderand indigoid dyesrdquo Journal of the Society of Dyers amp Colouristsvol 110 no 9 pp 273ndash277 1994

[4] L Burgio and R J H Clark ldquoLibrary of FT-raman spectraof pigments minerals pigment media and varnishesrdquo Spec-trochimica Acta Part A Molecular and Biomolecular Spec-troscopy vol 57 no 7 pp 1491ndash1521 2001

[5] F Casadio M Leona J R Lombardi and R Van Duyne ldquoIden-tification of organic colorants in fibers paints and glazes bysurface enhanced Raman spectroscopyrdquo Accounts of ChemicalResearch vol 43 no 6 pp 782ndash791 2010

[6] S Bruni V Guglielmi and F Pozzi ldquoHistorical organic dyesa surface-enhanced Raman scattering (SERS) spectral databaseon Ag Lee-Meisel colloids aggregated by NaClO

4rdquo Journal of

Raman Spectroscopy vol 42 no 6 pp 1267ndash1281 2011[7] P Colomban and D Mancini ldquoLacquerware pigment iden-

tification with fixed and mobile raman microspectrometersa potential technique to differentiate originalfake artworksrdquoArts vol 2 no 3 pp 111ndash123 2013

[8] A Zoppi C Lofrumento M Ricci E Cantisani T Fratini andE M Castellucci ldquoA novel piece of Minoan art in Italy the firstspectroscopic study of thewall paintings fromPhaistosrdquo Journalof Raman Spectroscopy vol 43 no 11 pp 1663ndash1670 2012

[9] L Burgio and R J H Clark ldquoComparative pigment analysis ofsix modern Egyptian papyri and an authentic one of the 13thcentury BC by Ramanmicroscopy other techniquesrdquo Journal ofRaman Spectroscopy vol 31 no 5 pp 395ndash401 2000

[10] D Bersani P P Lottici F Vignali and G Zanichelli ldquoA study ofmedieval illuminatedmanuscripts bymeans of portable Ramanequipmentsrdquo Journal of Raman Spectroscopy vol 37 no 10 pp1012ndash1018 2006

[11] A Zoppi C Lofrumento N F C Mendes and E M Castel-lucci ldquoMetal oxalates in paints a Raman investigation onthe relative reactivities of different pigments to oxalic acidsolutionsrdquo Analytical and Bioanalytical Chemistry vol 397 no2 pp 841ndash849 2010

[12] R Aroca Surface-Enhanced Vibrational Spectroscopy JohnWiley amp Sons Chichester UK 2006

[13] E C Le Ru and P G Etchegoin Principles of Surface-EnhancedRaman Spectroscopy Elsevier New York NY USA 2009

[14] A C Albrecht ldquoOn the theory of raman intensitiesrdquoThe Journalof Chemical Physics vol 34 no 5 p 1476 1961

[15] M Fleischmann P J Hendra and A J McQuillan ldquoRamanspectra of pyridine adsorbed at a silver electroderdquo ChemicalPhysics Letters vol 26 no 2 pp 163ndash166 1974

[16] P C Lee and D Meisel ldquoAdsorption and surface-enhancedRaman of dyes on silver and gold solsrdquo Journal of PhysicalChemistry vol 86 no 17 pp 3391ndash3395 1982

[17] E Vogel W Kiefer V Deckert and D Zeisel ldquoLaser-depositedsilver island films an investigation of their structure opticalproperties and SERS activityrdquo Journal of Raman Spectroscopyvol 29 no 8 pp 693ndash702 1998

[18] A V Whitney R P Van Duyne and F Casadio ldquoSilver islandfilms as substrate for Surface-Enhanced Raman Spectroscopy


(SERS) a methodological study on their application to Artistsrsquored dyestuffsrdquo in Advanced Environmental Chemical and Bio-logical Sensing Technologies III vol 5993 of Proceedings of SPIEBoston Mass USA October 2005

[19] ENalbant Esenturk andA RHightWalker ldquoSurface-enhancedRaman scattering spectroscopy via gold nanostarsrdquo Journal ofRaman Spectroscopy vol 40 no 1 pp 86ndash91 2009

[20] A Guerrero-Martınez S Barbosa I Pastoriza-Santos andL M Liz-Marzan ldquoNanostars shine bright for you colloidalsynthesis properties and applications of branched metallicnanoparticlesrdquo Current Opinion in Colloid amp Interface Sciencevol 16 no 2 pp 118ndash127 2011

[21] J-Q Hu Q Chen Z-X Xie et al ldquoA Simple and effective routefor the synthesis of crystalline silver nanorods and nanowiresrdquoAdvanced Functional Materials vol 14 no 2 pp 183ndash189 2004

[22] S E J Bell and S J Spence ldquoDisposable stable media forreproducible surface-enhanced Raman spectroscopyrdquo Analystvol 126 no 1 pp 1ndash3 2001

[23] M Leona J Stenger and E Ferloni ldquoApplication of surface-enhanced Raman scattering techniques to the ultrasensitiveidentification of natural dyes in works of artrdquo Journal of RamanSpectroscopy vol 37 no 10 pp 981ndash992 2006

[24] M Leona ldquoMicroanalysis of organic pigments and glazesin polychrome works of art by surface-enhanced resonanceRaman scatteringrdquo Proceedings of the National Academy ofSciences of the United States of America vol 106 no 35 pp14757ndash14762 2009

[25] A Idone M Gulmini A-I Henry et al ldquoSilver colloidal pastesfor dye analysis of reference and historical textile fibers usingdirect extractionless non-hydrolysis surface-enhanced Ramanspectroscopyrdquo Analyst vol 138 no 20 pp 5895ndash5903 2013

[26] Z Jurasekova E del Puerto G Bruno J V Garcıa-RamosS Sanchez-Cortes and C Domingo ldquoExtractionless non-hydrolysis surface-enhanced Raman spectroscopicdetection ofhistorical mordant dyes on textile fibersrdquo Journal of RamanSpectroscopy vol 41 no 11 pp 1455ndash1461 2010

[27] M Leona P Decuzzi T A Kubic G Gates and J R LombardildquoNondestructive identification of natural and synthetic organiccolorants in works of art by surface enhanced raman scatteringrdquoAnalytical Chemistry vol 83 no 11 pp 3990ndash3993 2011

[28] B Doherty B G Brunetti A Sgamellotti and C Miliani ldquoAdetachable SERS active cellulose film a minimally invasiveapproach to the study of painting lakesrdquo Journal of RamanSpectroscopy vol 42 no 11 pp 1932ndash1938 2011

[29] B Doherty F Presciutti A Sgamellotti B G Brunetti and CMiliani ldquoMonitoring of optimized SERS active gel substrates forpainting and paper substrates by unilateral NMR profilometryrdquoJournal of Raman Spectroscopy vol 45 no 11-12 pp 1153ndash11592014

[30] F Pozzi and M Leona ldquoSurface-enhanced raman spectroscopyin art and archaeologyrdquo Journal of Raman Spectroscopy vol 47no 1 pp 67ndash77 2016

[31] C Muehlethaler M Leona and J R Lombardi ldquoReview ofsurface enhanced Raman scattering applications in forensicsciencerdquo Analytical Chemistry vol 88 no 1 pp 152ndash169 2015

[32] H Schweppe ldquoIndigo and woadrdquo in Artistsrsquo Pigments AHandbook of Their History and Characteristics R L Feller EWest FitzHugh and A Roy Eds vol 3 pp 81ndash107 NationalGallery of Art Washington DC USA 1997

[33] Z Zhou G G Huang T Kato and Y Ozaki ldquoExperimentalparameters for the SERS of nitrate ion for label-free semi-quantitative detection of proteins and mechanism for proteins

to form SERS hot sites a SERS studyrdquo Journal of RamanSpectroscopy vol 42 no 9 pp 1713ndash1721 2011

[34] D Stulik D Miller H Khanjian et al Solvent Gels for theCleaning of Works of Art The Residue Question edited by VDorge Getty Publications 2004

[35] C Lofrumento M Ricci E Platania M Becucci and ECastellucci ldquoSERS detection of red organic dyes in Ag-agar gelrdquoJournal of Raman Spectroscopy vol 44 no 1 pp 47ndash54 2013

[36] E Platania J R Lombardi M Leona et al ldquoSuitability of Ag-agar gel for the microextraction of organic dyes on differentsubstrates the case study of wool silk printed cotton and apanel painting mock-uprdquo Journal of Raman Spectroscopy vol45 no 11-12 pp 1133ndash1139 2014

[37] E Platania C Lofrumento E Lottini E Azzaro M Ricciand M Becucci ldquoTailored micro-extraction method forRamanSERS detection of indigoids in ancient textilesrdquo Analyt-ical and Bioanalytical Chemistry vol 407 no 21 pp 6505ndash65142015

[38] C Lofrumento E Platania M Ricci C Mulana M Becucciand E M Castellucci ldquoThe SERS spectra of alizarin and itsionized species the contribution of the molecular resonance tothe spectral enhancementrdquo Journal of Molecular Structure vol1090 pp 98ndash106 2015

[39] C Lofrumento F Arci S Carlesi M Ricci E Castellucciand M Becucci ldquoSafranin-O dye in the ground state A studyby density functional theory Raman SERS and infrared spec-troscopyrdquo Spectrochimica Acta Part A Molecular and Biomolec-ular Spectroscopy vol 137 pp 677ndash684 2015

[40] S Gaurav and B Raja Digital Color Imaging Handbook Editedby G Sharma CRC Press New York NY USA 2002

[41] I T Shadi B Z Chowdhry M J Snowden and R WithnallldquoSemi-quantitative analysis of indigo by surface enhancedresonance Raman spectroscopy (SERRS) using silver colloidsrdquoSpectrochimica Acta Part A Molecular and Biomolecular Spec-troscopy vol 59 no 10 pp 2213ndash2220 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


are their very high tinting power (ie they are likely to befound in works of art in extremely low concentrations) andthe strong fluorescence signal generated by excitation withvisible light even when red or near red emitting laser is usedand that often obscures the inherently weakRaman scatteringsignal

A phenomenon that can significantly enhance the Ramansignal intensity is the surface-enhanced Raman scattering(SERS) effect The Raman scattering efficiency of moleculecan be enhanced when the molecule is effectively interactingwith a nanostructured metal surface and it is spatially con-fined within the electromagnetic field of the localized surfaceplasmon resonance (LSPR) of the nanostructured system[12 13] The localized plasma resonance furnishes a mean tofunnel electromagnetic energy in the proximity of the surfaceof the metallic nanoparticles (NPs) such that light scatteringphenomena of species adsorbed on the metallic surfacecan benefit from a substantial increase in the incomingand outgoing (diffused) electromagnetic fields SERS effectcan give enhancements of Raman signals greater than 8orders of magnitude Also SERS is a selective spectroscopictechnique as only molecules effectively interacting with theNPs show enhanced signals In our specific application ondyed textile fibers the SERS effect is observed only for thedye molecules and not for the substrate Finally it mustbe mentioned that the presence of metal NPs provides avery efficient way to quench fluorescence from the excitedmolecules thus removing a further serious limitation for theapplication of Raman spectroscopy to dye molecules [12 13]Therefore SERS is also very effective for discrimination ofmolecules of different nature present in artistic samples andthe enhancement of the signal enables us to detect even tracequantities of materials

Raman experiments can be carried out also at excitationfrequencies that are close to (or even resonant) the electronicexcitation frequency of the molecule under investigationIn that case we are dealing with the so-called ldquoresonanceRamanrdquo effect that produces much larger signals for spe-cific vibrational modes [14] Surface-enhanced [resonance]Raman scattering (SE[R]RS) is known to produce furtherenhancement of the Raman signal When the LSPR of theenhancing substrate is also in the proper energy region theSERRS enhancement factor is roughly the product of theenhancement factor for nonresonant SERS and the resonanceRaman spectrum intensification factor of the molecule

In order to improve the quality of the enhancementof the Raman scattering and the reproducibility of thespectra a wide class of nanostructured substrates such asrough electrodes [15] colloids [16] nanoisland films [17 18]nanostars [19 20] nanorods [21] and other nanocompositetechnological supports have been carried outThese advance-ments together with solid phase microextraction techniquesthat use synthetic and natural polymeric materials [22] haveallowed researchers in the field of cultural heritage to achieveconclusive identification of dyes from artworks with greatmolecular selectivity and specificity

The first applications SERS methods for dyes identifica-tion on textile fibers were still not enough sensitive to allownondestructive operations [23] A first significant step for

the increase of sensitivity of this method was represented bythe work of Leona [24] Very small (tens of micrometers)samples containing mordant dyes were treated with HFvapors in order to detach the dye from the mordant Then adroplet of colloidal solution containing silver NPs was addedto the sample and the dye was free to move and to bindthe NPs Other methods were developed that use a densedispersion of silver NPs directly on the fibers leading to anirreversible contamination of the original sample [25 26]An innovative and effective method for dyes microextractionfollowed by SERS dyes identification was recently presented[27] It was based on the use of common hydrogels basedon hydroxymethacrylates (contact lens blanks) loaded withwater organic solvents and chelating agents as extractionmedia Another method was presented based on the appli-cation to the sample surface of a cellulose film loaded withsilver NPs [28] The film dries in minutes after applicationand SERS spectra of anthraquinonic dyes present on thesurface were obtained with good sensitivityThe dimensionalchanges associated with the drying process possibly inducesmechanical stress on the sample surface leading to thedetachment of small fragments The film self-detaches or ispeeled off by the operator its formulation was optimizedin order to minimize the gel penetration in the samplesto obtain possibly its complete detachment and to limitthe detachment of small particles from the sample surface[29] Comprehensive reviews on the application of SERSto forensic science and cultural heritage have been alreadypublished [30 31]

This review will discuss the nondestructive microex-traction procedure we developed for dye extraction fromtextile fibers based on the use of an environmentally friendlynanocomposite hydrogel to provide a new tool for thoseresearchers who would like to embark on this kind ofwork using the SERS technique We will present the resultsobtained on mordant dyes (anthraquinones) and vat dyes(indigoids) used both in mockups prepared in the laboratoryand in ancient textiles

2 Materials and Methods

21 Dyeing of Textiles Dyes can be divided into three differ-ent categories (mordant vat and direct dyes) depending onthe procedure according to which they are applied to fabricsIn what follows we are providing information of the differentclasses of natural organic colorants used in textile dyeing inorder to clearly outline the approach we are using

Colorants named ldquomordant dyesrdquo do not have a strongchemical affinity for the textile fibers which therefore requirebeing treated prior to the dyeing stage involving a two-stepchemical reaction A solution of a ldquomordantrdquo is first usedto impregnate the fibers allowing the metal ion to becomecomplexed to appropriate functional groups in the structureof the textile During the dyeing process the colorant inter-acts with themordant-fiber complex via ionic and coordinatecovalent bonds to form insoluble brightly colored speciesThe complex thus formed within the fibers does not easilywash out of the textile and the resulting dyeing is thereforerelatively fast The mordant helped the dye bite onto the



4)2sdot12H2O)


2) were



2S2O4 Immer-







2S2O4ratio equal





HO OH

OH


Mordant (alum)

Alizarin

Al3+

O

O

Ominus


H

H

H

H

N

N

N

N

O

O

OH

HO

O2

Na2S2O4NaOH











2S2O41 2 ww water












Ethanol extraction

200 600 1000 1400 1800


Ram

an si

gnal

(au

)

00

02

04

06

08

10

(a)



200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(b)

Reference


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(c) (d)







200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)













200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



4)2sdot12H2O)


2) were



2S2O4 Immer-







2S2O4ratio equal





HO OH

OH


Mordant (alum)

Alizarin

Al3+

O

O

Ominus


H

H

H

H

N

N

N

N

O

O

OH

HO

O2

Na2S2O4NaOH











2S2O41 2 ww water












Ethanol extraction

200 600 1000 1400 1800


Ram

an si

gnal

(au

)

00

02

04

06

08

10

(a)



200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(b)

Reference


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(c) (d)







200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)













200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


HO OH

OH


Mordant (alum)

Alizarin

Al3+

O

O

Ominus


H

H

H

H

N

N

N

N

O

O

OH

HO

O2

Na2S2O4NaOH











2S2O41 2 ww water












Ethanol extraction

200 600 1000 1400 1800


Ram

an si

gnal

(au

)

00

02

04

06

08

10

(a)



200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(b)

Reference


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(c) (d)







200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)













200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




2S2O41 2 ww water












Ethanol extraction

200 600 1000 1400 1800


Ram

an si

gnal

(au

)

00

02

04

06

08

10

(a)



200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(b)

Reference


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(c) (d)







200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)













200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Ethanol extraction

200 600 1000 1400 1800


Ram

an si

gnal

(au

)

00

02

04

06

08

10

(a)



200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(b)

Reference


200 600 1000 1400 1800

Ram

an si

gnal

(au

)

00

02

04

06

08

10

(c) (d)







200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)













200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


200 600 1000 1400 1800


0

500

1000

1500

2000

SERS

Ram

an in

tens

ity

(a)

200 600 1000 1400 1800


0

1000

2000

3000

4000

5000

SERS

Ram

an in

tens

ity

(b)













200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


200 400 600 800 1000 1200 1400 1600 1800 20000

200

400

600

800


SERS

Ram

an in

tens

ity

200 400 600 800 1000 1200 1400 1600 1800 2000

591679 840 10381141

13421452

1483

1581

15911605

0

1000

2000

3000

4000


SERS

Ram

an in

tens

ity

(a)

(b)(c)



Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




Before After119871

lowast119886

lowast119887

lowastΔ119871

lowastΔ119886

lowastΔ119887

lowastΔ119864

lowast




4 Conclusions








Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






Competing Interests


Acknowledgments


References








4rdquo Journal of

















































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

review article microanalysis of organic pigments in

Documents