Investigation into the Use of Surface-Enhanced Raman Spectroscopy
(SERS) for Organic Dye Analysis
MASSACHUSETTS INSTITUTE
OF ECHNOLOGy
by
by
JUN 08 2015
Sally Lin
LIBRARIES
SUBMITTED TO THE DEPARTMENT OF MATERIALS SCIENCE AND ENGINEERING
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF SCIENCE IN MATERIALS SCIENCE AND ENGINEERING
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2015
2015 Sally Lin. All rights reserved.
The author hereby grants to MIT permission to reproduce
and to distribute publicly paper and electronic
copies of this thesis document in whole or in part
in any medium now known or hereafter created.
Signature of Author:
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Department of Materials Science and Engineering
May 1st, 2015
Certified by:
Signature redacted
V f/
/
Jeffrey C. Grossman
Professor of Materials Science and Engineering
Thesis Supervisor
Certified by:
Sian ature redacted
Nicola Ferralis
Res arch SAentist MateriI Science and Engineering
Thesis Supervisor
/n
-
Accepted by:
Sig nature redacted
V
Geoffrey S.D. Beach
'0
Professor of Materials Science and Engineering
Chairman, Committee for Undergraduate Students
'
2
Investigation into the Use of Surface-Enhanced Raman Spectroscopy
(SERS) for Organic Dye Analysis
by
Sally Lin
Submitted to the Department of Materials Science and Engineering
on May 1st, 2015 in Partial Fulfillment of the
Requirements for the Degree of Bachelor of Science in
Materials Science and Engineering
ABSTRACT
In art conservation, color is essential to understanding a society's culture and history-as
an indicator of beauty, status, religion, and more-but has a tendency to fade and diminish
over time. Analytical techniques, particularly that of pigment identification, can reveal the
artifact's original color and appearance and give new insights to an artist's intentions,
techniques, date of creation, and more. However, most identification procedures are
invasive and destroy the samples in the process. Surface-enhanced Raman spectroscopy
(SERS) has recently been identified as a technique that is minimally invasive and also
solves the issue of fluorescence that is found in many other techniques. In this paper, a
specific SERS procedure has been developed for the identification of yellow organic dyes
from 18th century Japanese Woodblock prints. Several SERS spectra of nine dyes both in
solution and applied on artist paper have also been documented in hopes of assisting with
pigment identification in the future.
Thesis Supervisor: Jeffrey C. Grossman
Title: Professor of Materials Science and Engineering
Thesis Supervisor: Nicola Ferralis
Title: Research Scientist, Materials Science and Engineering
3
4
Table of Contents
1 . L ist o f F ig u re s ....................................................................................................................................................
7
2 . L is t o f T a b le s ......................................................................................................................................................
9
3 . In tro d u ctio n ....................................................................................................................................................
11
4 . L ite ra tu re R e vie w .........................................................................................................................................
16
4.1. Ram an Spectroscopy ....................................................................................................................
16
4.2. Surface-Enhanced Ram an Spectroscopy (SERS) .........................................................
19
4.3. SERS Successes with Organic Dyes ....................................................................................
22
4.4. Organic Dyes from 18t" Century Japanese Woodblock Prints ..............
24
5. Materials and Methods ...............................................................................................................................
25
5.1. Dye Specim ens and Gold Colloids .......................................................................................
25
5.2. Dye Solution and Colloid Sample Preparation ..............................................................
26
5.3. Dye Applied on Paper and Colloid Sample Preparation ...........................................
27
5.4. SERS Setup and Experimental Procedure .......................................................................
27
6. Results and Discussion ..............................................................................................................................
29
6 .1 . O p tica l Im a g in g ...............................................................................................................................
29
6.2. Raman Spectra of Dye Solutions with Aggregated Colloid ......................................
32
6.3. Ram an Spectra of Dye Solutions with Non-Aggregated Colloid ............................
37
6.4. Ram an Spectra of Dye Solutions with Varying Acquisition Tim es ....................... 37
6.5. Raman Spectra of Dyes Applied on Paper .......................................................................
38
6.6. Predictive Analysis: Structure to Spectra .......................................................................
40
7 . C o n c lu s io n s .....................................................................................................................................................
43
8 . A ck n o w le d g e m en ts .....................................................................................................................................
46
5
9 . R e fe r e n c e s .......................................................................................................................................................
48
Appendix A: Dye Components and Classifications ............................................................................
50
Appendix B: Optimal SERS Procedure for the Collection of Organic Yellow Dyes
from 181 Century Japanese Woodblock Prints ................................................................
51
Appendix C: SERS Spectra for Dye Solutions with Aggregated Colloid ....................................
52
Appendix D: SERS Spectra for Dye Solutions with Non-Aggregated Colloid...........................
58
Appendix E: SERS Spectra for Dyes Applied on Paper ....................................................................
60
6
1. List of Figures
Figure 1. Raphael's "Portrait of a Cardinal" 1510 ...............................................................................
12
Figure 2. W inslow Hom er's "For to Be a Farm er's Boy" 1887 ..........................................................
13
Figure 3. Rayleigh and Raman scattering types based on molecular energy states ............ 17
Figure 4. Normal Raman spectroscopy compared to SERS ...........................................................
18
Figure 5. Enhanced Raman scattering due to the use of metal substrates in SERS ............. 20
Figure 6: SERS comparison of an artifact fiber sample
and a reference m adder-dyed fiber ....................................................................................
23
Figure 7: Sam ple dye solution and dye applied on paper ..............................................................
26
Figure 8: Optical images of representative dye-colloid samples ................................................
30
Figure 9: Optical image of ukon dye-colloid at 100x magnification and SERS spectrum
of ukon dye-colloid aim ed at a yellow area ......................................................................
31
Figure 10: N ine sam ple dye solutions .....................................................................................................
32
Figure 11: SERS spectrum of pure aggregated colloid ....................................................................
33
Figure 12: SERS spectrum of ukon dye with aggregated colloid ................................................
34
Figure 13: SERS spectrum of purpurin ..................................................................................................
36
Figure 14: SERS spectra of pure aggregated colloid .........................................................................
38
Figure 15: Representative SERS spectra of kihada dye solution and dyed fibers ................ 39
Figure 16: Representative SERS spectra of ukon dye solution and dyed fibers ..................
40
Figure 17: Chem ical Structure of chrom ophores ...............................................................................
41
Figure 18: Molecules that correspond to the peaks 581 cm-1, 721 cm', and 1429 cm1 ...... 42
Figure 19: Raman spectra of acetone (Sacher Lasertechnick) .....................................................
7
43
Figure C1: SERS spectra of enju dye with aggregated colloid ........................................................
53
Figure C2: SERS spectra of kariyasu dye with aggregated colloid .............................................
53
Figure C3: SERS spectra of kihada dye with aggregated colloid ...................................................
54
Figure C4: SERS spectra of kuchinashi dye with aggregated colloid ..........................................
54
Figure CS: SERS spectra of te-o dye with aggregated colloid ........................................................
55
Figure C6: SERS spectra of ukon dye with aggregated colloid ......................................................
55
Figure C7: SERS spectra of yamamomo dye with aggregated colloid ...........................................
56
Figure C8: SERS spectra of woren dye with aggregated colloid ......................................................
56
Figure C9: SERS spectra of akane dye with aggregated colloid .......................................................
57
Figure D1: SERS spectra of enju dye with non-aggregated colloid .............................................
58
Figure D2: SERS spectra of kariyasu dye with non-aggregated colloid ....................................
58
Figure D3: SERS spectra of kihada dye with non-aggregated colloid ........................................
59
Figure D4: SERS spectra of akane dye with non-aggregated colloid .........................................
59
Figure E1: SERS spectra of enju dye applied on artist paper with aggregated colloid .......... 60
Figure E2: SERS spectra of kariyasu dye applied on artist paper with aggregated colloid . 60
Figure E3: SERS spectra of kihada dye applied on artist paper with aggregated colloid ..... 61
Figure E4: SERS spectra of kuchinashi dye applied on artist paper
with aggregated c o llo id ...............................................................................................................
Figure ES: SERS spectra of te-o dye applied on artist paper with aggregated colloid .....
61
62
Figure E6: SERS spectra of ukon dye applied on artist paper with aggregated colloid ........ 62
Figure E7: SERS spectra of yamamomo dye applied on artist paper
w ith aggregated colloid ..............................................................................................................
8
63
2. List of Tables
Table 1: Dyes under analysis and sample type in possession ......................................................
25
Table 2: Com m on peak values for each dye solution ......................................................................
35
Table 3: Comparison of common peaks found in spectrum of experimental akane
and its chrom ophore purpurin from literature ..............................................................
9
36
10
3. Introduction
The restoration and preservation of art is essential to understanding and preserving
a society's culture and history. For art conservationists, such artifacts not only exist for
appreciation and beauty, but also reveal a population's lifestyles, rituals, trade routes,
available substances, and much more. In particular, an artifact's color can speak volumes.
For example, in many cultures throughout history and modern times, vibrant and striking
colors reflect one's hierarchical status and social class (Figure 1). In modern times, dyes
have even become widely used in food, drugs, and cosmetics, to name a few. However,
colors fade over time and under prolonged exposure to light and the elements, and the
original appearance may be lost (Figure 2). Successful analysis of artworks can give an idea
of the artifact's original color and appearance and give new insights to an artist's original
intentions, techniques, and date of creation. These analytical methods also have the
potential to uncover forgeries and falsifications. Successful identification and conservation
techniques have great promise for a variety of applications.
Conservation research itself strives to date artifacts and analyze artists' materials
and techniques. According to Pozzi, the identification of historical dyes in art materials is
one of the most challenging of chemical investigations due to a variety of reasons (Pozzi,
2011). First, colorants in artifacts are often incorporated in complex matrices of paint
layers, textile fibers, and binding media, and are typically used in diluted concentrations.
Second, organic materials also undergo chemical degradation, and can change an original
molecular structure in its entirety. Finally, analytical research is often invasive. Ideally,
techniques are conducted without interfering with the actual artwork, but it is often times
impossible to do without extracting a sample of the artifact for analysis. There is also
11
Figure 1. Raphael's "Portrait of a Cardinal" 1510. In Medieval
Europe, wearing bright red colors was an exclusive right of the
nobility. This was in part due to the difficulty in preparing
bright colors from natural sources, that only the wealthy could
hope to afford it.
difficulty in transferring identification techniques from the laboratory to the actual works
of art under consideration.
According to Brosseau et al., the ideal analytical tool for dye identification must
meet the following criteria: it must be minimally destructive, highly diagnostic, sensitive,
and can probe a broad range of samples in a variety of mediums and matrices (Brosseau et
al., 2009). Current techniques developed for colorant identification in historical artifacts
12
Figure 2. Winslow Homer's "For to Be a Farmer's Boy" 1887. (A) Current
appearance of the watercolor painting. (B) Digital simulation of the original
color according to X-ray fluorescence spectroscopy and FTIR analysis. The
pigments Homer originally used to paint the sky, chrome yellow and pink
madder, faded over time with exposure to light. A new interpretation of the
piece revolves around the contemplation of a peasant worker at sunset of a
hard day.
13
fail to meet these standards in various ways: UV-vis spectroscopy often produces rather
featureless spectra for dyes of similar hues; FTIR places constraints on sample uniformity,
thickness, and dilution that is difficult to meet due to complex sample matrices;
3DFluorescence spectroscopy lacks an elemental signature; and high performance liquid
chromatography (HPLC), while the most promising, requires a large sample size of 0.5 - 5
mm in diameter (Centeno & Shamir, 2008; Soltzberg, 2012; Harroun et al., 2011; Casadio et
al., 2010). This issue with invasive and many times destructive analysis is of particular
importance and thus current research strives to develop the most minimally invasive
techniques possible.
Luckily, Raman spectroscopy, which utilizes the phenomenon of light photons
scattering after interacting with matter, partially addresses this problem. Although this
technique still requires a sample to be taken, the sample may be miniscule (a matter of
microns) and the process itself is not destructive, so the same small sample can be reused
for additional analysis. Because of this, Raman spectroscopy has become a commonplace
technique in many conservation centers. However, there are some important downsides to
Raman spectroscopy. Namely, that it is inherently a weak process and can be easily masked
by fluorescence interference (Caycedo 2012). As a result, Raman spectroscopy has typically
been applied to inorganic, or synthetic, dyes, which exhibit less fluorescence than organic,
or natural, dyes do (Pozzi, 2011). Unfortunately, this limits the technique to works from the
mid-191 century onward, when the first synthetic dyes were made. For artifacts created
prior to that point, Raman spectroscopy is not as successful.
These drawbacks are potentially addressed by utilizing surface-enhanced Raman
spectroscopy (SERS). SERS makes use of a metal substrate to increase scatter and enhance
14
the detection of the signal. One trade-off is that the samples analyzed using SERS are
destroyed, although the samples themselves can be even more miniscule than what regular
Raman spectroscopy requires. Still, recent research has shown the usefulness of SERS on
the identification and analysis of both organic and inorganic dyes. For instance, in 2008
Van Elslande et al. conducted the first direct SERS study of an actual archeological pigment
containing purpurin, and three years later, Pozzi found success in identifying yellow dye
found in wool threads from an evacuation site in the Libyan Sahara, dating back to the
ancient Garamantian period (Pozzi 2011). Several other experiments have yielded
successes in identifying colorants in historical pastel artworks, watercolors, paintings, and
textile fibers (Harroun et al., 2011). Even so, the use of SERS on artifacts is still under
development, and has not yet been applied to many molecules. There exists a need to fill
the database of reference materials with these Raman spectra.
The goal of this project is to develop SERS as a technique to analyze certain organic
dyes from 18th century Japan. These dyes were applied as very thin layers onto woodblocks
and then printed on paper; due to this thinness, extracted samples cannot be analyzed
successfully through the usual techniques. Because SERS can be applied on miniscule
samples as well as counter the strong fluorescence these dyes usually exhibit, this project
centered around two goals: (1) developing an optimal SERS methodology for organic dyes
that is easily reproducible and exhibits greater identification success than existing
techniques and (2) building a database of reproducible Raman spectra for several reference
dyes. The hope is to, with further research, successfully apply this methodology on a
variety of actual artifacts in the art conservation case.
15
4. Literature Review
This section begins with an overview of the theories behind Raman spectroscopy
and SERS. Following that is a discussion of successes with SERS pigment identification and
the decision to analyze the 18t" century organic dyes in question.
4.1 Raman Spectroscopy
Raman spectroscopy is a microscopic technique that makes use of light interacting
with matter. Although useful, it is an inherently weak process and is unsuccessfully applied
on natural dyes due to fluorescent interference.
When light interacts with matter, the photons are absorbed, scattered, or simply
passed through the medium. For absorption to occur, the energy of the incident photon
must correspond to the energy gap between a molecule's ground state and excited state. If
so, the photon is absorbed, and this absorbance can be measured with spectroscopy. On the
other hand, if a photon scatters, the energy does not need to correspond to the energy gap.
Various different types of scatter may occur this way. Rayleigh, or elastic, scattering occurs
if the light photons simply distorts and polarizes the electron clouds of the molecule
(Figure 2). This places the molecule in an unstable state, and it re-radiates the energy,
although only in small changes in frequency. In most cases, Rayleigh scattering is the
dominant process. In another vein, Raman, or inelastic scattering, occurs when the photons
affect nuclear motion, leading to an increase in the molecule's vibration amplitude and
larger energy transfer (Figure 3). As only one in 106 to 108 molecules are scattered this
way, Raman spectroscopy is inherently a very weak process (Smith & Dent, 2005).
16
A h.
Virtual states
Energy
I_____
-~
_________1~I_______ Vibrational states
___
Rayleigh
scattering
Stokes
Raman
scattering
Anti-Stokes
Raman
scattering
Figure 3. Rayleigh and Raman scattering types based on molecular energy states.
Rayleigh scattering is an elastic distortion of the electron cloud and is the dominant
process. Raman scattering (Stokes and anti-Stokes) affect nuclear motion and is
inelastic, and occurs in only 1 in 106 to 108 molecules (Raman Scattering, 2014).
Still, Raman spectroscopy is one of the most ideal techniques for molecule
identification and has been proven successful in various cases. In particular, unlike other
techniques, Raman spectroscopy is non-destructive. Although a sample usually must be
taken in order to perform an analysis, the sample may be miniscule, requires little to no
preparation, and once the analysis is finished, the same sample may be used in another
technique. Moreover, with sufficient preparation, Raman spectroscopy can even be
performed in-situ, which has great implications (Caycedo, 2012). Many cases of successful
identification through Raman spectroscopy have occurred due to the above reasons. For
example, using Raman spectroscopy, Caycedo successfully identified a number of inorganic
dyes from the Schweppe collection, both in pure solution and applied on textile fibers, and
Frano et al. successfully identified the inorganic pigment Vermilion in Isaac Barre, an oil
17
painting by Sir Joshua Reynolds (Caycedo, 2012; Frano et al., 2014). The non-destructive
aspect, miniscule sample size, and ability to be performed in-situ make Raman
spectroscopy a most promising technique proven to be applicable to several works of art.
Still, barriers exist that limit the successes of Raman spectroscopy. While it is indeed
useful for identifying inorganic pigments, it faces difficulty identifying organic dyes. This is
because natural dyes are often applied in very dilute concentrations yet exhibit strong
fluorescence, which is unseen in synthetic dyes. Because Raman scattering is weak, this
fluorescent interference easily covers a material's Raman spectrum and limits its
identification abilities (Figure 4). Thus, although Raman spectroscopy is promising, the
main obstacles in utilizing normal Raman spectroscopy are its inherently weak signals and
fluorescent interference. Luckily, the invention of surface-enhanced Raman spectroscopy,
or SERS, has addressed both of these issues.
Naskd(
t
fFaz
.
F
"MMN
Aulma
1%
nldi
_Ms
al
ONN/
Figure 4. Normal Raman spectroscopy (black) compared to SERS (red). In this case of Flavazine L.
at a 785nm laser, the normal Raman spectrum is covered by fluorescent interference. With SERS,
more distinct peaks can be seen and then used in successful pigment identification (Caycedo, 2012).
18
4.2 Surface-EnhancedRaman Spectroscopy (SERS)
Surface-Enhanced Raman Spectroscopy is as the name implies: the technique makes
use of a metal substrate to magnify the inherently weak Raman signal and reveal more
distinct peaks in its Raman spectrum. It addresses the fluorescence issue while keeping the
advantages of simplicity and minimal sample preparation as seen in normal Raman
spectroscopy, and is currently one of the most promising techniques for art conservation.
The theory behind SERS builds upon that of normal Raman spectroscopy. In SERS,
the sample as an anlayte is first adsorbed onto a roughened metal substrate. The
roughened metal substrates contain electrons covering its surface so that when light hits
the surface, the electrons begin to oscillate and become surface plasmons. These surface
plasmons have specific resonance frequencies at which they most efficiently absorb and
scatter energy, depending on the nature of the metal. In particular, silver and gold have
oscillation frequencies in the visible spectrum and are thus useful for SERS (Smith, 2005).
The roughened surface ensures that light will be scattered, rather than simply absorbed as
with a smooth, parallel surface. The scattering-to-absorbing ratio and type of roughening of
the metal substrate are both factors that may affect the outcome of the spectra.
Once the analyte is adsorbed onto the metal substrate and surface plasmons form,
the energy from the plasmons transfers to the adsorbed molecules. Some of the energy is
used to affect nuclear motion on the sample, thereby initiating the Raman process on the
molecule. The rest of the energy is transferred back to the plasmon and scattered from the
surface under shifted wavelengths (Figure 5). The result is a greatly enhanced signal-by a
factor of 107 to 109 for pyridine and even up to 101 in some cases (McNay et al., 2011). If
the incident light radiation is similar to the plasmon frequency and molecular vibration, the
19
Raman Scattering
X 106 - 1014
10' I'
Analyte
Figure 5. Enhanced Raman scattering due to the use of metal substrates
in SERS (SERS, 2007).
resonance and therefore overall signal will increase even more. The resulting enhancement
is enough to cover the fluorescent interference exhibited in normal Raman spectroscopy
and renders that problem irrelevant. Furthermore, this enhancement increases the
reliability and sensitivity with which a sample of low concentration is identified, as any
contamination is easily recognized in the spectrum (McNay et al., 2011). In this manner,
SERS is able to reveal a much clearer, reliable, and more distinct Raman spectrum than
would otherwise.
To better explain the enhancement phenomenon, two competing theories exist. The
first, and most widely accepted, relies on electromagnetic enhancement and is generally
described as above. The second relies on a charge-transfer mechanism, focusing on an
electron-hole pair interaction. In this case, the analyte molecule from the sample is bound
chemically to the conduction band of a noble metal surface. Energy is transferred in order
to form these new bonds, and the resultant electronic states promote Raman scattering.
While this charge-transfer mechanism is certainly evidenced in the process, it is commonly
20
believed that electromagnetic enhancement plays a larger part in this phenomenon, since
this bonding between the analyte and substrate does not occur in all cases (Pozzi, 2011).
Many potential substrates may be used for SERS, but the ideal surface species
depends on the nature of the surface as well as the analyte itself. Typically, metal
substrates are either silver or gold colloids. Silver surfaces will generally be oxidized silver
ions (Ag+) due to the presence of oxygen, and will be highly reactive. The risk here is that
the substrate may not be reproducible or have a good lifetime. Gold surfaces, on the other
hand, are much more stable and do not run the same risks. The advantage to using colloids
is that the suspensions are fluids and much more compatible with certain analyte
procedures. Furthermore, both gold and silver colloids are easy to prepare, reasonably
stable, and have similar absorbance wavelengths--gold absorbs at 500nm, silver at 532 nm
excitations (McNay et al., 2011). Other substrates that have been researched include metal
covered polystyrene spheres, silver tollen mirrors, and silver nanoisland films, but none
have worked as well as the silver and gold colloids (Leona et al., 2006). Specifically, Leona
et al., Caycedo, and others champion sodium citrate-reduced silver colloids as the most
successful substrate (Leona et al., 2006; Caycedo, 2012). Ultimately, the choice of metal
substrate is dictated by the desired plasmon resonance frequency-for the purposes of this
experiment, silver and gold frequencies are ideal as their plasmon frequencies fall within
the visible spectral range (Pozzi, 2011).
An ideal SERS technique depends on a number of factors, in addition to a good
substrate as mentioned above. The analyte must adsorb on the metal substrate effectively
and on a large enough cross-section. The shape and size of the metal nanoparticles must be
taken into account. The molecular excitation levels must be controlled to prevent surface
21
photodecomposition as might occur with high laser power and long acquisition times
(Smith & Dent, 2005). The spectrum obtained must be reproducible and robust, and the
substrate should have an adequately long lifetime and strong enhancement effects. Finally,
to produce reliable quantitative results, many events must be averaged and then compared
with a standard to view any changes.
There are, of course, certain disadvantages to the use of SERS that prevent it from
becoming as widespread a technique. First, because the sample must adsorb onto a metal
substrate, the technique is invasive and destructive, and must be applied in the laboratory.
This disadvantage is somewhat offset by the huge enhancement of the Raman signalbecause of this increased signal, even smaller samples can be used-on the scale of
microns. Secondly, the SERS spectrum may differ greatly from the normal Raman spectrum.
New bands and peaks may form or others may disappear, depending on the sample in
question (Caycedo, 2012). Work still remains in this area. Still, SERS is a technique of
choice in the art conservation community because of its high sensitivity, minimally invasive
nature, little preparation, and overall simplicity.
4.3 SERS Successes with OrganicDyes
Since ancient times, dyes were derived from natural sources such as plants-roots,
berries, wood, leaves-and organisms-lichens, insects, fungi. The colors obtained are
mainly blue, red, yellow, with other colors formed through a mixture of these primary
three.
Although the SERS technique was discovered over four decades ago, it has only
recently been applied to the identification of cultural heritage objects in art and
22
archaeology. Past research has shown successes with SERS identification of red lake
pigments, most extensively with madder (a red color), although also with other dyes such
as Tyrian purple or select dye collections (Bruni, 2009; Brosseau, 2009; Caycedo 2012).
The Raman spectra obtained in these experiments have been tested on actual historic
works of art including paintings, pastels, and archaeological artifacts with promising
results, as seen in Figure 6. Other dyes in multiple complexes have also been documented,
such as paints mixed with binders and mordants, house paint and wallpaper, etc. (Harroun,
2011).
The yellow pigment is the next logical choice for absorption spectra documentation.
As mentioned, the most common dyes used are red, yellow, and blue. As the red pigment
has been studied most extensively, both yellow and blue are of particular interest. Few
studies have been documented on either, and in the case of the yellow pigments, the
Rww
"
I
'
A
Figure 6: SERS comparison of (1) a fiber sample from the artifact
"The maiden's companion signals to the hunters" and (2) a reference
madder-dyed fiber (Casadio, 2010).
23
fluorescence is stronger than that of the blue pigments. As a result, SERS would be most
useful to apply on the yellow dyes due to its ability to quench the fluorescence.
4.4 OrganicDyesfrom 18th CenturyJapanese Woodblock Prints
With the choice of yellow pigments decided as thus, the question of which dyes to
choose was determined as a matter of convenience and interest. The Museum of Fine Arts
in Boston, Massachusetts, has a renowned Asian Conservation center. Of current interest
are Japanese woodblock prints created prior to 1856, after which the first synthetic dyes
quickly overtook the market.
Traditional eastern print-making utilizes pigments mixed with water. The dyes are
applied onto woodblocks in very thin layers then printed onto artist paper. The colors dye
the paper fibers and are fused together much as the process with textiles is (Salter, 2002).
This is in contrast with Western techniques, which utilizes pigments mixed with oil, so that
the pigments stay adsorbed on the surface rather than fusing with the fibers.
The organic yellow dyes in this study were prepared at the museum from original
recipes from the time period of interest. The remainder of this paper details the
development of SERS as a reproducible technique to identify and document these dyes of
interest and determine potential predictive relationships between structure and spectra
for future identification applications.
24
5. Materials and Methods
5.1 Dye Specimens and Gold Colloids
Organic yellow dye specimens were prepared at and obtained from the Scientific
Conservation office in the Museum of Fine Arts in Boston. The specimens were of two
types: (1) Nine dye solutions prepared as cold water extracts (20 mL of filtered tap water
mixed with 10 mL of the raw colorant, allowed to set overnight) and (2) seven of the nine
dye solutions applied on artist paper (Table 1, Figure 7). Between the two dyes that did not
have an applied paper form was one madder (red) dye-named "akane"-with which to
compare against literature-documented reference dyes. A comprehensive list of the dyes,
their plant/mineral sources, chromophores, and more can be found in Appendix A.
PELCO® NanoXact TM Gold Colloids were ordered from Ted Pella, Inc. They had a size
of 60 nm (2.29x10 10 particles/mL), were supplied at 1x concentration in Milli-Q water, and
were titanic acid capped. When not in use, the gold colloids were wrapped in aluminum foil
and stored at 4C. Gold colloids were chosen over the more common silver colloids due to a
desire for a less reactive material as well as microscopy limitations.
A summary of the procedure as detailed in the next sections is found in Appendix B.
Table 1: Dyes under analysis and sample type in possession
(solution, fiber, or both).
Jaans
Nam
Souton Fier orot
Both
Both
Both
Both
Both
Both
Both
Solution
Solution
Enju
Kariyasu
Kihada
Kuchinashi
Te-o
Ukon
Yamamomo
Woren
Akane
25
Figure 7: Sample dye solution and dye applied on paper. Both are of
kariyasu dye, named "rice plant" in English.
5.2 Dye Solution and Colloid Sample Preparation
To prepare SERS samples from the dye solutions, the following procedure was
followed. First, for this section "aggregated" and "non-aggregated" colloid solutions were
tested as a measure of comparison. Non-aggregated colloids were taken straight from the
supplied solution. Aggregated colloids were created through the following procedure: 2 mL
of supplied gold colloid was centrifuged for 25 min at 3500 rpm. The resulting top clear
layer of water was pipetted out and discarded. Once the colloids were ready, 10 uL of dye
solution was placed onto a glass slide. 10 uL of non-aggregated or aggregated gold colloid
was then added onto the drop and stirred. The mixed drop was left to dry under a constant
stream of nitrogen gas.
26
5.3 Dye Applied on Paperand Colloid Sample Preparation
To prepare SERS samples from the dyes applied on paper, the following procedure
was followed. First, the colloids were aggregated in a manner identical to that for the dye
solutions. Next, single fibers were extracted from the dyes applied on paper and placed on a
glass slide. A drop of methanol was added on top of the fiber to loosen the dye, and left to
dry for a few minutes. The procedure for the dye solutions was then implemented-10 uL
of aggregated colloid was added on top of the fiber and left to dry under a constant stream
of nitrogen gas.
5.4 SERS Setup and Experimental Procedure
A Horiba LabRam HR800 microscope with a 633 nm (red) laser was used, with
grating at 600. Power was optimized at 0.1% of the maximum and at 100x magnification.
Using HORIBA Scientific's LabSpec 5 software, optical images were taken of each sample at
10x and 100x magnification. Raman (absorption) spectra were taken at a range of -50 cm-1
to 2000 cm- 1 and at a variety of acquisitions, including 5 accumulations of 1 second each, 5
accumulations of 3 seconds each, 10 accumulations of 1 second each, 10 accumulations of 3
seconds each, 20 accumulations of 1 second each, and 20 accumulations of 3 seconds each.
When taking spectra, it was best to focus on areas of high dye and colloid concentration,
often with greater weight to an abundance of colloid. Spectra should be taken within a few
minutes of drying, to avoid contamination from airborne particles.
For the dye solutions, prior to collecting any dye spectra, a Raman spectrum of pure
aggregated or non-aggregated colloid was collected on each new batch of dye collection.
This is to normalize each dye solution spectra by manually subtracting this pure colloid
27
spectrum from the dye solution spectra using Microsoft Excel (the colloid spectrum was
often multiplied by an appropriate constant). Common peaks were then identified from the
normalized dye spectra.
For the dyes applied on paper, Raman spectra of both pure aggregated colloid and
aggregated colloid applied on pure paper were collected prior to each new batch of dye
collection. These spectra were manually subtracted from spectra of dyes applied on paper
and again common peaks were then identified.
In the following section, the resulting common peaks are identified as potential
reference spectra for the dyes by comparing the dye solutions, the dyes applied on paper,
and spectra from literature, in the case of madder. Once these peaks are identified, a
predictive analysis is performed to determine the relationship between structure and
spectra.
28
6. Results and Discussion
6.1 Optical Imaging
Optical images of each sample were taken at magnification 10x and 100x. Figure 8
contains representative images of the dye-colloid samples. Generally, the ability to take
consistent and distinct spectra correlated to what could be seen during optical imaging.
For best spectra, the laser was aimed at areas of high dye and colloid concentration,
with heavier weight to areas of colloid clusters. This is best seen in the 100x magnification
view (Figure 8-B and 8-D): darker splotches are dye molecules while the bright, reflective
spots are gold colloid particles. Dye molecules were often clustered together and it was
best to aim for those clusters, provided a cluster of colloid particles was also present. For
example, in Figure 8-B, there is an abundance of dye molecules as well as colloid particles
and strong spectra can be obtained from any area with clusters of colloid. In contrast, in
Figure 8-D, there is a large cluster of dye molecules but not many clusters of gold
nanoparticles, and consequently it was difficult to obtain good spectra. Moreover, some
dyes, most notably the ukon and somewhat the kihada and woren, fluoresced strongly and
their yellow color could be seen under the 100x magnification (Figure 9-top). In these
cases, it was best to avoid the yellow spots, as even with SERS the spectrum would be
masked by the strong yellow fluorescence (Figure 9-bottom). However, there were plenty
of ideal areas without yellow fluorescence that gave strong Raman spectra. Finally, with
other dyes, particularly the kuchinashi and akane, there was difficulty in finding large
enough dye molecules under 100x magnification. This could be reflected by their lighter insolution color and thinness of the dye solutions (Figure 10). In general, the best spectra
was taken by aiming the laser at clusters of high dye and colloid concentrations, with
29
- ---
Figure 8: Optical images of representative dye-colloid samples: (A) enju dye solution with
aggregated colloid at 10x magnification, (B) woren dye solution with aggregated colloid at 100x
magnification, (C) kariyasu dye solution with non-aggregated colloid at 10x magnification, (D)
kariyasu dye solution with non-aggregated colloid at 100x magnification, (E) fibers of kuchinashi
dye applied on paper with aggregated colloid at 10x magnification, and (F) a single fiber of
kuchinashi dye applied on paper with aggregated colloid at 100x magnification.
30
- __ -1
special attention paid to issues that correlated with physical properties of the dyes.
There are a few differences in comparing the optical images of dyes with
aggregated and non-aggregated colloids, and with fibers of applied dye. For aggregated and
non-aggregated samples, while the 10x magnification images are largely the same, the 100x
magnification images show noticeably less colloid in the non-aggregated samples. This
3000
2500
2000
W
C 1500
C
1000
500
0
0Z 4 Cn Or4Q %-qfL
M r*-
C
W
A
m
,
,
-4W
L
Raman Shift (cmA-1)
Figure 9: (Top) Optical image of ukon dye-colloid at 100x magnification. A slight yellow tinge can
be seen in the clump in the middle and upper left. (Bottom) SERS spectrum of ukon dye-colloid
aimed at a yellow clump. The high intensity here overwhelms the spectrum and produces no peaks.
The yellow observed is an indicator of strong fluorescence and should be avoided when taking
spectra.
31
Figure 10: Nine dye sample solutions. From left to right, the dyes are: enju,
kariyasu, kihada, kuchinashi, te-o, ukon, yamamomo, akane, and woren. The
different shades of yellow correlate with some observed findings, e.g. te-o,
ukon, and yamamomo have relatively thicker solutions.
made it much harder to even detect a Raman spectrum, much less a consistent and reliable
one. For the fibers of dye applied on paper, the samples clearly looked different and it was
much more difficult to focus on the fibers due to the differences in depth. However, it was
still possible to obtain reliable spectra by focusing the laser on the clusters of colloid
present on the fibers as seen in Figure 8-F. The best practices as discussed for the dye
solution with aggregated colloid samples can still be applied to non-aggregated colloid and
dyed fibers, with these adjustments in mind.
6.2 Raman Spectra of Dye Solutions with Aggregated Colloid
Using the mentioned procedures and best practices, fairly consistent spectra were
obtained for dye solution with aggregated colloid samples. In this section, the
reproducibility and accuracy of the experimental spectra are analyzed, including
32
comparisons with literature.
First, SERS was taken with pure dye solutions (no colloid). As expected, no
distinguishable spectra appeared. This was true for all samples-by performing SERS on an
area absent of colloids, no distinguishable spectra will appear.
Next, a pure colloid spectrum was taken at the beginning of every run. Figure 11
shows a colloid spectrum, typical of both aggregated and non-aggregated colloid. This pure
colloid spectrum has been consistently and reliably observed throughout the experiment.
From spectra of all other samples, these pure colloid spectra were manually subtracted
(after multiplying by a constant) to obtain the resulting normalized distinctive SERS
spectra.
For the nine dye solutions, their spectra were taken and normalized as mentioned.
SERS spectra for all nine dyes can be found in Appendix C; the spectrum for ukon is shown
in Figure 12 as a representative example. The consistency and reproducibility of ukon can
180
160
140
120
100
80
r
C4
oo
M M
-W
O
V
O
WLAino
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LA
d
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M
t
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a
b
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4
-
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-..
-
60
1-4
I
-
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4
MA
N
U)
.4,4
Raman Shift (cmA-1)
Figure 11: SERS spectrum of pure aggregated colloid, taken at 633nm with 20
accumulations of 3s each.
33
I
600
S00
400
300
-5-3_2
10-31
10-32
C
200
2-
100
00
~t- -4 OM Mrfl 0 O 0
0
f%V-4
C0MP%
0 r4W
00 r4
O
0
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.
Mv f- 9-4 LA
v0I-4 WR0
f", 00 00
V0 M
r
w
81 f4 f" -4L0
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0
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-4 r-4INr4 C4
V-4 V-4 r4 - 49
M
-4
-4
IH
V3
1..
4y.4
-
NP-W94U 0
04 7-4 V-4 y.4 T.4 V-4
Raman Shift (cm^-1)
Figure 12: SERS spectrum of ukon dye with aggregated colloid at two different spots, laser of
633nm with each spot taken at 5 accumulations of 3s and 10 accumulations of 3s, with one 20
accumulations of 3s.
be seen, and a number of distinct peaks can be separated from the baselines.
Most dye spectra were fairly consistent and the spectra in Appendix C are
representative of numerous trials for each dye. Kariyasu, kihada, te-o, and ukon had the
most consistent and reproducible spectra while enju, yamamomo, and akane dyes were
somewhat less reproducible. Kuchinashi was the least consistent and reliable, and there
was difficulty obtaining similar shapes on different runs. Why this is true is unknown with
this small dye sample size and may be explored in future research.
Distinct peaks could be fairly confidently determined for most of the dyes except for
the kuchinashi dye. Approximate common peak values observed from the many trials are
recorded in Table 2. As seen, each dye has a distinct combination of peaks that can be used
to distinguish it from the others.
To verify the accuracy of this procedure, the spectra of the sole red dye, akane, was
34
Table 2: Common peak values for each dye solution with a 633nm laser taken
over a number of trials.
Enju
582, 654, 720, 996, 1303, 1428
Kariyasu
580, 723, 908, 1238, 1375, 1426, 1508
Kihada
450, 579, 716, 903, 1304, 1436, 1628
Kuchinashi*
366, 544, 580, 615, 732, 818, 1058, 1154, 1192
Te-o
314, 436, 618, 745, 921, 1413, 1577
Ukon
474, 555, 720, 941, 1014, 1080, 1205, 1316
Yamamomo
594, 659, 742, 818, 1060, 1432, 1548
Woren
372, 659, 760, 866, 1331, 1483, 1616
Akane
424,482,611,680, 1027, 1143, 1214, 1309, 1370, 1428, 1619
*Keeping in mind that this dye's spectrum is inconsistent.
compared to that of literature. The English name for akane is Japanese madder. As
mentioned, madder has been extensively researched and its family of Raman spectra has
been recorded in a number of databases. Akane contains the chromophore purpurin, whose
reference Raman spectra can be seen in Figure 13. Table 3 compares the observable peaks
of this literature purpurin spectra with that of this experimental akane spectrum. We can
see that many peaks appear in approximately the same locations and the overall shape of
the two spectra are quite similar-both generally take the form of skewed bell shape
curves centering around 1330 cm 1 or 1350 cm 1 (the representative spectra of akane can
be found in Appendix C). However, a number of peaks do differ, and it is important to note
that the two spectra were prepared and executed under different conditions, which likely
affected the peaks distribution. As such, judging by this comparison of experimental akane
and literature purpurin, it can be said that this SERS methodology provides generally
accurate Raman spectra, although some specific peaks may differ.
35
1349
i
T
I
1600
'
'
I
1200
I
I
'
400
800
Wavenumber (cm *)
Figure 13: SERS spectrum of purpurin prepared on AgFONs fabricated
with 390 nm diameter SiO 2 spheres and taken with 15s acquisition
with excitation at 632.8nm (Whitney, 2007). Spectra are not expected
to align completely due to the differing preparation methods.
Table 3: Comparison of common peaks found in spectrum of experimental
akane and its chromo hore ur urin from literature Whitne , 2006).
424
433
482
611
680
--
-1027
-1143
1214
468
-657
708
800
859
967
-1102
1135
1201
1309
1370
1428
1619
-1349
-1614
36
6.3 Raman Spectra of Dye Solutions with Non-Aggregated Colloid
To develop an ideal procedure for the use of SERS on pigment identification, the
colloid itself was also experimented with to obtain the best method. As mentioned, the
colloid itself is a fundamental element of SERS. Enough colloid must be present for the dye
molecules to adhere onto and enhance the Raman signal. Thus, two colloid preparations
were tested: non-aggregated colloids (the colloid solution exactly as was supplied) and
aggregated colloids (centrifuged solution). Various concentrations of non-aggregated
colloid solution were tested on six of the nine dye solutions, but as seen in the optical
imaging section in Figure 8-D, these colloid particles were generally not abundant enough
to provide consistently strong or reproducible spectra. The spectra that resulted from
these non-aggregated samples exhibited few to no consistent peaks and could not compare
to the strength of the spectra from the aggregated colloid samples (see Appendix D for
examples of non-aggregated spectra). However, it can be noted for consistency's sake that
many of the peaks that could be observed were similar to those found in the aggregated
colloid samples (i.e. spectra from Appendix C), particularly that of kariyasu. Thus, it was
determined that supplied colloid in itself could not produce reliable and distinguishable
spectra, but the aggregated colloid could.
6.4 Raman Spectra of Dye Solutions with Varying Acquisition Times
To further develop an ideal SERS procedure, acquisition times were varied. Each dye
solution was tested with acquisition combinations of: 5 accumulations of is, 5
accumulations of 3s, 10 accumulations of Is, 10 accumulations of 3s, 20 accumulations of
is, and 20 accumulations of 3s. Acquiring for too long was undesirable, as prolonged
37
exposure to the laser could potentially burn the area of the sample under laser. It was
determined that for the pure colloid sample-aggregated or non-aggregated-20
accumulations of 3s was best, as the pure colloid samples tended to be relatively more
noisy (Figure 14). For all the other dyes, either spectra taken at 10 accumulations of 3s or
20 accumulations of 3s was usually enough to match the smoothness of the pure colloid
samples taken at 20 accumulations of 3s and result in distinguishable peaks (see any of the
spectra in Appendix C).
300
250
200
F-
150
A-
ksN
5-1
\
-
s-1
----
100
10
-
C
,J
10-3
20-1
20-3
50
- --
r -4~.
Mcrl~
---------
---
------
_
--------------
OW0 r 'WV-4LnM -4l A W %W-0 4L MM
Lnlf MO~O
%
0 --
0
W-1 4y4
- 4 4 V- -4 r4 q 9-4
O
Dn0qtMM
4
-4
-4
-4 4
l
r
flV-
n
W
Raman Shift (cmA-1)
Figure 14: SERS spectra of pure aggregated colloid with laser of 633nm, taken at the same spot
with varying acquisition times: 5 accumulations of 1 s, 10 accumulations of is and 3s, and 20
accumulations of is and 3s.
6.5 Raman Spectra of Dyes Applied on Paper
To further test the reliability of the resulting spectra and to check the feasibility of
applying this technique on actual artist mediums (with the eventual goal being to apply
SERS onto artifacts), SERS was performed on dyes applied to artist paper. Similar to the dye
38
solutions where a pure aggregated colloid SERS sample was taken prior to each run, SERS
on blank artist paper fibers with aggregated colloid were taken prior to each run of applied
paper samples. These spectra were then manually subtracted from the spectra of dyed
fibers. In these cases, the shape of the blank fiber-colloid spectra appeared very similar to
that of pure aggregated colloid-the fibers themselves seemed not to exhibit any distinct
peaks.
Appendix E contains spectra for the seven dyes applied on paper. The spectra
themselves are fairly consistent and reproducible. However, when comparing with spectra
from the dye solutions, the consistency between the solutions and paper do not match as
well as was hoped. To illustrate, Figure 15 overlays representative spectra of the kihada
solution and dyed fiber while Figure 16 does the same for ukon. While the general shapes
of the spectra are similar, the peaks do not match up perfectly or approach the same
distinction from the baseline. As a result, the accuracy of this SERS technique as an
250
200
)%150
\
-
100
Dye
Solution
Dyed
Fibers
50
0
WI9-u-, w8"Lf
j
oo
M fl-
IPT " t-lO
Ur4r-SCM
T-
"1 V4
"-
~'r -4r iy4r ir ir i
O
Raman Shift (cm^-1)
Figure 15: Representative SERS spectra of kihada dye solution and dyed fibers with laser of
633nm, both acquired at 20 accumulations of 3s.
39
application to the use of dyes on paper or other mediums rather than in pure solution form
may have issues that still need to be addressed.
400
350
300A
250
A
~~V
. Dye
VI
C 200
Solution
C
Dyed
Fibers
150
100
50
0
V-4 11
V-4 r46 V46
4
V4 v.
V-4 V-4
v.9 v6V4 6V-4 -I V.4 V-4 r-4
Raman Shift (cm^-1)
Figure 16: Representative SERS spectra of ukon dye solution and dyed fibers with laser of
633nm, both acquired at 10 accumulations of 3s.
6.6 PredictiveAnalysis: Structure to Spectra
The final aspect of this project was to determine whether a predictive relationship
could be found between the structure of the dyes and their SERS spectra. The goal is to be
able to, from the spectra of an unknown but pure sample, determine which specific dye it
contains. To that end, dyes enju, kariyasu, and yamamomo were analyzed for potential
predictive aspects.
First, a number of considerations must be kept in mind. There are numerous
combinations of dyes, peaks, and chemical bonds that may be considered, but in this
development stage only one subset was analyzed. Dyes enju, kariyasu, and yamamomo
40
were chosen as the subset because they were the only dyes among the samples that had
some similarity in their components-all three belonged to the flavonoid colorant
classification, making the three simpler to analyze and compare. However, the accuracy of
this comparison must be qualified. This analysis was based on the chemical formulas for
the chromophores of each dye given in Appendix A. The exact structure and composition of
each dye is unknown, as the distributions of the chromophores were not specified.
Figure 17 shows the chemical structure of the chromophores for dyes enju,
kariyasu, and yamamomo. As seen, the structures of these chromophores are largely the
same, with differences only in the number and placement of hydroxyl groups and a
functional group for genistin. This similarity is expected and the next step is to determine
whether these differences and similarities correspond to their resulting spectra.
OH
HO
OH
HO
0
OH
OH
HO
OH
OH
0
0
Myricetin
Morin
0H
HO
OH
OH
OH
HO
H 0t
0
OH
OH
OH
Quercetin
Genistin
Figure 17: Chemical Structure of chromophores for kariyasu (morin), yamamomo
(myricetin and quercetin) and enju (quercetin and genistin).
41
Upon observing the peaks noted for enju, kariyasu, and yamamomo, it was
determined that all three dyes share common peaks at 581 cm-1 and 1429 cm-1.
Additionally, enju and yamamomo shared a peak at 657 cm- 1 and enju and kariyasu shared
a peak at 721 cm-1. Once these common peaks were established, the next step was to
determine what chemical structure these common peaks might correspond to. This was
done through an online database of vibrational energy values provided by the National
Institute of Standards and Technology (NIST, 2011). Absorption peaks can arise from the
vibration of one or more bonds. This phenomenon can take the form of a stretch between
two atoms, a compression or bending of a bond, a torque, or a rocking motion, and each
type is represented by a unique vibration. Each such bond is specific to a particular
molecule, which the database was able to provide.
It was determined that the 581 cm- 1 peak corresponded to the molecule C 6 H 4 (OH) 2+,
the 1429 cm-1 peak to C 6 HsO, and the 721 cm-1 to C 3 H 6 0 (acetone), all of which are
structures common to that of the chromophores mentioned previously (Figure 18). No
molecule could be found whose bonds exhibited a vibrational energy of 657 cm-1 that fit the
dyes' chemical structure.
To confirm that these vibrational energies did reflect their respective peaks, a
0
OH
|
OH
C6 H 4 (OH)z+
C6 HsO
Acetone
Figure 18: Molecules containing bonds whose vibrational energies correspond
to the peaks 581 cm-1 (C 6 H 4 (OH)
2 +),
721 cnr 1 (C 6 H 5 0), and 1429 cm- 1 (acetone).
42
Raman spectra of acetone was found and shown in Figure 19. As seen, acetone does contain
a distinct peak at 1440 cm-1, which is quite close to the 1428 cm-1 experimental peak value.
Although Raman spectra could not be found for the other two molecular structures, this
confirmation of acetone alone already gives confidence to this predictive analysis.
790
aX
--
Acetne
1440
1727
re tI:6 a
IIea
1W- I
0
A
200
4X
600
a0
1000
12i0
140
1&0
1&W
2000
Figure 19: Raman spectra of acetone (Sacher Lasertechnick, 2014).
To further increase confidence, the peaks of the other six sample dyes were also
compared. It was found that for the most part, if their chemical structures contained any of
the molecules in Figure 18, their Raman spectra had corresponding peaks in the locations
as mentioned. Thus, for these three dyes-enju, kariyasu, and yamamomo-it is fairly
reasonable to assume that if one were to find a peak of an unknown sample at 581 cm-1,
721 cm-1, and 1428 cm-1, the dye is either enju or kariyasu; and if one were to find a peak of
an unknown sample at 581 cm-1, 657 cm-1, and 1428 cm-1, the dye is either enju or
yamamomo.
The procedure developed here serves as the basis for future extrapolation of its
predictive capabilities onto other dyes. Although an actual identification on an unknown
Raman spectrum was not tested, the process is simple to perform in the future and holds
great potential, keeping in mind certain unavoidable constraints as mentioned.
43
8. Conclusions
The goal of this project was to develop SERS as a reproducible technique to identify
and document nine yellow organic dyes from woodblock prints in 18th century Japan in
hopes of initializing a reference database for such dyes. To that end, the resulting spectra
exhibited general consistency and reproducibility and even a predictive aspect, but faced
difficulties in providing specific peak distinction.
The procedure that was developed through this project can assist future research in
obtaining the best SERS spectra on these specific dye samples as well as on other similar
dyes, though to what extent this procedure can be applied remains to be seen in future
research. However, it is clear that some best practices can be followed that have not been
detailed in previous literature, such as using optical imaging to aim at areas of high colloid
concentration and avoid areas of fluorescence, or yellow color in this case. The spectra
obtained in this project using this method are best used as general references for
identification of these yellow dyes. Fortunately, even with this approximation, a predictive
process relating chemical structure with SERS absorption peaks could be developed for the
dyes enju, kariyasu, and yamamomo, with the potential to extend to other dyes.
With further research and additional testing under varying conditions, more specific
peaks and predictive rules can be accurately and reliably produced. For instance, the
supplied colloid may be more effective if silica-coated shells were added on the gold
nanoparticles. The advantage of silica-coated shells in SERS is that the coating prevents
over-aggregation of the nanoparticles and ensures that enough nanoparticle surface is
exposed for the dye molecules to adhere to. PELCO offers BioPure TM gold colloids capped
with a 20nm thick silica shell, and this would be a straightforward additional project to
44
complete. Moreover, as mentioned before, further predictive analysis can be performed on
the six dyes that had not yet been analyzed. An actual identification of an unknown Raman
spectra can also be conducted following this predictive analysis method that was
developed for enju, kariyasu, and yamamomo dyes. These are rather quick and
straightforward additional research areas that may be completed with time. With these
adjustments and more, it may be possible to build a more specific and accurate database
for these dyes.
The development of SERS as a consistent and reproducible technique and creation
of a predictive database with these target dyes can have a large effect on real world
applications such as art conservation, identification of forgeries, understanding historic art
techniques, pinpointing available materials, and more. As seen, there are currently still
issues with the SERS technique that need to be resolved before it can be applied to actual
artifacts from the time period in question. However, this technique has potential, and with
further research, a reliable SERS technique and procedure can certainly be developed to
create a much more specific and comprehensive database of dyes.
45
9. Acknowledgements
I would like to thank Professor Jeffrey C. Grossman for supervising my work, Professor
Nicola Ferralis who taught me the SERS technique and constantly assisted my analysis,
Brent Keller for helping me implement the procedure, and the entire Grossman Group for
letting me use their laboratory and materials.
I would also like to thank the Boston Museum of Fine Arts (MFA) for helping form this
project and for providing materials. In particular, Mr. Richard Newman and Ms. Michele
Derrick have assisted me much in the formulation of this project.
Finally, I would like to thank Professor Lorna Gibson for connecting me with the MFA in the
first place, and for being a wonderful advisor throughout all my years at MIT.
46
47
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McNay, G., Eustace, D., Smith, W., Faulds, K., & Graham, D. (2011). Surface-Enhanced Raman
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Energy Value. Retrieved April 21, 2015 from
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http://webbook.nist.gov/chemistry/vib-ser.html
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Pozzi, F., van den Berg, K. J., Fiedler, I., Casadio, F. (2014). A systematic analysis of red lake
pigments in French Impressionist and Post-Impressionist paintings by surfaceenhanced Raman spectroscopy (SERS). Journalof Raman Spectroscopy, 45, 11191126.
Raman Scattering, University of Cambridge. (2014). Retrieved February 27, 2015, from
http://www.doitpoms.ac.uk/tlplib/raman/raman-scattering.php
Raphael. (1510). Portrait of a Cardinal [Online image]. Retrieved February 27,2015 from
http://en.wikipedia.org/wiki/Portrait-of_a_Cardinal._%28Raphael%29
Raman Scattering, University of Cambridge. (2014). Retrieved February 27, 2015, from
http://www.doitpoms.ac.uk/tlplib/raman/raman-scattering.php
Sacher Lasertechnik. Raman Spectroscopy: Acetone Detection and Identification, (2014).
Retrieved April 28, 2015, from http://www.sacherlaser.com/applications/overview/raman-spectroscopy/acetone.html
Salter, Rebecca. JapaneseWoodblock Printing. Honolulu: U of Hawai'i, 2002.
SERS, Pennsylvania State University. (2007). Retrieved February 27, 2015, from
http://research.chem.psu.edu/lxjgroup/pagel6.html
Smith, E., & Dent, G. (2005). Modern Raman spectroscopy: A practicalapproach. Hoboken,
NJ: J. Wiley.
Soltzberg, L. (2012). 3D Fluorescence Characterization of Synthetic Organic Dyes. American
Journalof Analytical Chemistry, 3, 622-631.
Whitney, A., Casadio, F., & Duyne, R. (2006). An innovative surface-enhanced Raman
spectroscopy (SERS) method for the identification of six historical red lakes and
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Whitney, A., Casadio, F., & Duyne, R. (2007). Identification and Characterization of Artists'
Red Dyes and Their Mixtures by Surface-Enhanced Raman Spectroscopy. Applied
Spectroscopy, 61(9), 994-1000.
49
Appendix A: Dye Components and Classifications
Name Japanese
Solution
(English),
_Fiber?
Enju (Pagoda Tree)
Both
a
Colorant -
neaClassification
Styphnolobium
japonicus;Sophora
Criohr
W t
oe
rutin, quercetin, sophoricosid,
Flavonoid
kaempferol, genistin
Flavonoid
morin (ossajin, pimaferin)
Alkaloid
Berberine
Gardenin; Crocin
japonica
Kariyasu (Rice
Both
(r0
Miscanthus tinctorius;
Miscanthussenensis
Plant)
Kihada (Yellowood;
Amur Cork Tree)
Both
Phellodendron
amurense
Kuchinashi
Both
Gardeniajasminoides;
Carotenoid;
(Gardenia)
Gardeniaaugusta
Flavonoid sch
Te-o, Kusha Shio
(Gamboge)
Garciniatrees; Garcinia
Ganit
Garcinia
rGca
Xanthones and
Hydroxanthones
Bh
morella
gambogic acid, isogambogic acid,
guttiferic acid; morelloflavon;
morellic acid, isomorellic acid;
morellin, isomorellin
Both
Curcuma longo;
Curcuma domestica;
Curcuma aromatica
Curcumin
Diferuloylmethane; Demethoxy
curcumin; Bisdemethoxy curcumin
(Mountain Peach)
Both
Myrica rubia
Flavonoid
myricetin, quercetin, kaempferol
Woren (Gold
Solution
Coptis trifolia
Alkaloid
berberine, coptin
Ukon (Turmeric)
Yamamomo
Thread)
Akane (Madder)
strong yellow
fluorescence
bright
fluorescence
strong yellow
fluorescence
Solution
Rubia akane
Anthraquinones
purpurin, pseudopurpurin
munjistmn
Appendix B: Optimal SERS Procedure for the Collection of Organic Yellow Dyes from 18th
Century Japanese Woodblock Prints
1. Colloid Preparation
a. Aggregated Colloids:
i. Centrifuge 2 mL of supplied gold colloid for 25 min at 3500 rpm
ii. Pipette out and discard the top clear layer of water
b. Non-Aggregated Colloids:
i. Use straight from the supplied solution.
2. Sample Preparation
a. Dye Solution
i. Place10 uL of dye solution onto a glass slide
ii. Add 10 uL of aggregated or non-aggregated gold colloid onto the drop
iii. Stir and let dry under a constant stream of nitrogen gas
b. Dye Applied on Paper
i. Extract single fibers from the samples and place on a glass slide
ii. Add a drop of methanol to loosen the dye from the fiber, let dry
iii. Add 10 uL of aggregated gold colloid onto the fiber
iv. Stir and let dry under a constant stream of nitrogen gas
3. SERS Technique
a. Setup
i. Laser to 633 nm (red)
ii. Grating at 600
51
iii. Power at 0.1% of the maximum
iv. Acquisition range of -50 cm
1 to
2000 cm
1
v. Acquisitions of 10 or 20 accumulations of 3s
b. Collect spectra of pure aggregated and/or non-aggregated colloid and/or
aggregated colloid on paper fibers at the beginning of each new batch
c.
Collect spectra of dye solution/paper fiber samples
d. Normalize these sample spectra by subtracting the spectra from (b)
i.
e.
May need to multiply the spectra from (b) by a constant
Tips
i. Take spectra within minutes of sample drying
ii.
Focus on areas of high dye and colloid concentration, with greater
weight to an abundance of colloid
iii. Avoid areas with yellow tinge, which indicates high fluorescence
52
Appendix C: SERS Spectra for Dye Solutions with Aggregated Colloid
900
800
700
-
600
10-31
400
20-3_1
300
20-3_2
T,
200
100
0
0
QeJr.r~
AO
' in
eq
M - eJ
en
m
r
'D
tt
LnQ
Ln Ln
Ln
'
00
rrZID
N
"0%
Lo n)
con 'rrnt eon qm-- "rI
NIN mn
0
MO
Ln Ln
"
to
toF-
Raman Shift (cmA-1)
Figure Cl: SERS spectra of enju dye with aggregated colloid at two different spots, laser of 633nm
with each spot taken at 10 accumulations of 3s and 20 accumulations of 3s.
700
500
400
5-3_1
300-5-3_2
10-31
200
10-32
100
20-3
0
v- 4 4 4 4 Z
~WW4 T-4 T4 W4V-
r~-ID
w.U
I-i i
OQm
r, .4
, -I LA
v-4 v-
49-4v-4
v
4
Raman Shift (cmA-1)
Figure C2: SERS spectra of kariyasu dye with aggregated colloid at two different spots, laser of
633nm with each spot taken at 5 accumulations of 3s, 10 accumulations of 3s, and one at 20
accumulations of 3s.
53
600
Soo
10-3_1
C
---
10-3_2
-
20-3_1
C 200
20-32
100
0
Wn
ChO~
M
v-YI%4U
OON 0
r4Go C4 W
%DI 0
M q4 Lfl M~ M V4
"
W44UV"
Raman Shift (cmA-1)
Figure C3: SERS spectra of kihada dye with aggregated colloid at two different spots, laser of
633nm with each spot taken at 10 accumulations of 3s and 20 accumulations of 3s.
4000
3500
/11,
-*1
3000
2500
2000
.---10-31
-10-32
1500
10-3_3
1000
___10-3_4
500
Raman Shift (cm^-1)
Figure C4: SERS spectra of kuchinashi dye with aggregated colloid at four different spots, laser of
633nm with each spot taken at both 10 accumulations of 3s. Spectra for this dye were not consistent.
54
170
150
ISO
130y
110
-5-3_1
-
5-3_2
C
70
10-3_1
10-3_2
0
50
20-3
30
10
~O
~
fr. .ra~~~~
00 0 -4v-4 " -4r-4 q- 3-4LTA- v- 0k 04 WNr
v-4
v--4
v
Raman Shift (cm^-1)
Figure C4: SERS spectra of te-o dye with aggregated colloid at two different spots, laser of 633nm
with each spot taken at 5 accumulations of 3s, 10 accumulations of 3s, and one at 20 accumulations
of 3s.
600
S00
400
-5-3
1
-5-3_2
300
--
10-3_1
10-3_2
200
/
20-3
100
"
n
WNW
r' '0
U
-
---
N
-4'.
0
~
00
_
-
---
N'.
-4
Mr% C%4 W 2 Lf M
N C" M q* R '.LA LM 000 M~R-,-
_
LAM~
AnC 1
N
V-4
y
ID- DAP
----
------
-----
_
N
w-4 LA
0o
0
rI'
V1 9 C" W
0
N
V~ Mm
'V-4 LA
N
V-4
A
-------- --
A
0
Raman Shift (cmA-1)
Figure C6: SERS spectra of ukon dye with aggregated colloid at two different spots, laser of 633nm
with each spot taken at 5 accumulations of 3s and 10 accumulations of 3s, with one 20
accumulations of 3s.
55
2000
1800
1600
1400
1200
5-3_1
1000
5-3_2
800
10-3_1
10-3_2
400
200
0
M
r4
M
-
v v LA~ un
u
W W
t
N
M W Mi M~ M~ 00 9
V4
4 V-4 N N
.4W
I -4
M M
4rq 94v .4 4 -4W-1TAT-4 4
-n )n Wt
V-4
N
Raman Shift (cmAft1)
Figure C7: SERS spectra of yamamomo dye with aggregated colloid at two different spots, laser of
633nm with each spot taken at 5 accumulations of 3s and 10 accumulations of 3s.
350
300
p200
I
19
5-3_2
10
10-31
//
so
-10-3_2
r
~
~
20-3_1
20-3_2
0
r-.
00
N4
.
MV
Mn
0
~N'
O
U
In
vUi
M
tn
mm
r
-4"Lnl
11%WW
Ln Ln
in
P, rNN
4 0
%%Il. 00 o MAT-40 9 V-V-4 4 v~ 1-43 in
nWt V"qV4
MWv
m
V4
rO'.0
N
ILn
m
;
M
r-
r4
W4
q-4
N4
v
m
w
"w0
o
WV-04U1
-4
r4
v-4
V-4
1
Raman Shfit (cm^-1)
Figure C8: SERS spectra of woren dye with aggregated colloid at two different spots, laser of
633nm with each spot taken at 5 accumulations of 3s, 10 accumulations of 3s, and 20
accumulations of 3s.
56
6000
5000
4000
-5-3_1
3000
1
210-3
-10-3
2000
2
120-31
20-3_2
1000
eNI'
al Mrl T
0
-r"r4t
N M M
T
-Cr -
t r
L
Ln Ln r 00
Go
N7 a r4T4r4rt "gr AL
4
--
1
-
-4 r4
-4
-4
r4
.
-
I-4 - 4 -4 -
-
Mt W-0
N ko
Raman Shift (cm^-1)
Figure C9: SERS spectra of akane dye with aggregated colloid at two different spots, laser of 633nm
with each spot taken at 5 accumulations of 3s, 10 accumulations of 3s, and 20 accumulations of 3s.
57
Appendix D: SERS Spectra for Dye Solutions with Non-Aggregated Colloid
2000
1800
1600
1400
1200
C1000
C
-
10-3j
W
......
--
10-3-2
10-3_3
600
-- 10-34
400200
0
~
-~-
r
%4DO
00 C" t- N~
-------
--
-
NW
M
On
L
N
-------
tr. TU &n~O
r1% V4%W
fOf10'
9
T"4
-
~ ,gf 0 ~'r A4D-U
4 i1 4
q-4
4 i4
y-4 to 0Q1M
-
vIY
y
Y
Iy
Raman Shift (cmA-1)
Figure D1: SERS spectra of enju dye with non-aggregated colloid at four different spots, laser of
633nm with each spot taken at 10 accumulations of 3s.
350
300
250
{
~200
100
10-33
50
0
ra'ei
C44
M~
-" ,-
cr
LM L4 vLl N4
tI ,-4 W- WI NI N
a~
"MaWIL~
00 IA12. A~ Lm 2 v .PWI 101 M n C4
r
Raman Shift (cn^-1)
Figure D2: SERS spectra of kariyasu dye with non-aggregated colloid at three different spots, laser
of 633nm with each spot taken at 10 accumulations of 3s. Spectra for this kariyasu dye was most
consistent, but ultimately unreliable as the sole consistent non-aggregated dye sample.
58
2000
1800
1600
1400
1200
10-33
C 1000
0
-10-32
800
41-.
0-3_1
600
400
200
0
----------
-P-% r-4
W 0 q Go
t"
4
q WD V- &n0
UI~X w5 L r -4w g LAl &
C"arn
0"
-r
r- r- )ln M00 W0
M~ fr- V-4 Ln0q 00 (M^
C Dn ov-OLf0R
1M
iV-4 v1
4V4
v.
1-1
4e
1r4 W-4Ir V-4 V.1 4
v
4 W-
-L
W- .4 v4 v41 W4 W-4
Raman Shift (cmAi1
Figure D3: SERS spectra of kihada dye with non-aggregated colloid at three different spots, laser of
633nm with each spot taken at 10 accumulations of 3s.
900
800
600
600
-U;
C
C
10-3_
10-33
fv
10-3_3
300
200
100
0
0 v 00 N
UD R LM. M
~IlN
W~.4~
P,
0 v W0 N WD 0 v M% -I4 01 M MW N WD 0 v
v-I LAO
N r- - W 0 S M8 "NFl -4
r- r- 00r-NIfD"CO0%
WO W-I U1 M~ MV rl v.q
Ln L
v-
v.4
r4O~~
vv-4
(
v.4 v.4 v-4
P
v-4
v-4
v-4
v.4
v4
v4
44v-I
. I v4
l-
Raman Shift (cmAl1
Figure D4: SERS spectra of akane dye with non-aggregated colloid at three different spots, laser of
633nm with each spot taken at 10 accumulations of 3s.
59
Appendix E: SERS Spectra for Dyes Applied on Paper
300
250
200
C 150
10-3 1
10-32
100
20-31
-20-3_2
so
0
1%
0
V00
rVD
rn
-4ifLO Menr.%9-4L
M~~~~~~~~~~~~~~~~~
M
Ln n r %.000 M
W4 V4 NN
MV
00
OD 0
Z A LM Wn Wf P%1 %.
Ch f" Pn v-4 Ln IM M~ 00 MI L10
N na !9
V44 ANr - na"P
-4
in 0
N
tO*
v-4 v-4 v4 v-4 r.4 v-4 r-4 R- 4r -4v-4
4 v- 1 v-4 9-4
4v.
Raman Shift (cmA-1)
Figure El: SERS spectra of enju dye applied on artist paper with aggregated colloid, laser of 633nm
taken at two different spots at 10 accumulations of 3s and 20 accumulations of 3s.
350
300
250
-5-3
j~200
C
-10-1
~1so
10-3
100
-3
50
W w4
Inp
m r-v-40n AN0r W0O Zmmn -4 &m MM r,-4
8
tOO0~
CO C" P~. Ni
r M C"~
fl
D
O
in in
~in
i-4i
r C 0o0
V' 4* r% -4 ADl
? q oLen
v4 01
R DMM N
U~
" -4
~r4
4
V-4
v.
r -4 Me n im Winnt
V4 v-4
V-4 T.-4
v-4
.4 9-4 v.4 v
4
v-49v-4
Raman Shift (cm^-1)
Figure E2: SERS spectra of kariyasu dye applied on artist paper with aggregated colloid, laser of
633nm with varied acquisitions at 5 accumulations of 3s, 10 accumulations of is and 3s, and 20
accumulations of is and 3s.
60
350
300
250
200
150
-10-3
20-3
100
20-3
50
0
C"
oMrM%
WtCL4n
LflO
i
N
r,
wZV,4
M
V-4rLn
Wfl~r
N411r
T4 -4 V-4
riq4
M
T4-4-4 -4T-y
i 4
T-
M
No
-4U2
4
Raman Shift (cm^-1)
Figure E3: SERS spectra of kihada dye applied on artist paper with aggregated colloid, laser of
633nm with varied acquisitions at 5 accumulations of 3s, 10 accumulations of Is and 3s, and 20
accumulations of is and 3s.
300
250
100
10-1
10-3
100
-20-1
0
P
y
~
W0gwQ y4 LfQ
N Or
OO gD
M r- %W4 LAfm
M
- - 4 y- - . 4 v4 v - - -4 V-4 %-I r-4
-
W-
Raman Shift (cmA-1)
Figure E4: SERS spectra of kuchinashi dye applied on artist paper with aggregated colloid, laser of
633nm with varied acquisitions at 5 accumulations of 3s, 10 accumulations of is and 3s, and 20
accumulations of is and 3s.
61
-----
900
800
700
600
-20-3
1
20-3 2
200
100
0
1*% 94~toA
4Ln
Mr
-
Lfl4t
W
I-V4Ln
M8
V- 4 -4 -4 4 V4 -4 -I " r41-4 v4 v-4 *-4 "4 v-4 v-4
Raman Shift (cmA-1)
Figure E5: SERS spectra of te-o dye applied on artist paper with aggregated colloid, laser of
633nm taken at two different spots at 10 accumulations of 3s and 20 accumulations of 3s.
800
500
-
400
-
600
-10-1
V
10-3
30020-1
2--20-3
100
--
0
Raman Shift (cmA-1)
Figure E6: SERS spectra of ukon dye applied on artist paper with aggregated colloid, laser of
633nm with varied acquisitions at 5 accumulations of 3s, 10 accumulations of is and 3s, and 20
accumulations of is and 3s.
62
120
100
-10-31
--
10-3-2
20-31
40
20-32
60
20
V-4 M N VI
eq W
M
fl-V-4Ln M4 Wf
r-
W
Uo N4~ rz
8
33 r WW
v--4
v-
Y
..
y4v- v- v-4 v-4 v4 "q T - 4 vq4 -4
Raman Shift (cmA-1)
Figure E7: SERS spectra of yamamomo dye applied on artist paper with aggregated colloid, laser of
633nm taken at two different spots at 10 accumulations of 3s and 20 accumulations of 3s.
63