Application eBook
Raman spectroscopyContents
Semiconductor

Material science

Evaluation of SiC semiconductors …………………… 4

Evaluating the materials
in rechargeable batteries …………………………………… 13

Evaluation of polymorphism in SiC
Distribution analysis of 3C-SiC on 4H-SiC wafer

Evaluation of the cathodeEvaluating carrier density in SiC(Tips) Atmospheric shielding cell and electrochemistry cell

Non-destructive depth proﬁle measurement of 4H-SiC
epitaxial layer

Evaluation of the binder

Evaluation of component in a multi-layer structureof SiC wafer using DSF and MCR analysis(Tips) DSF (Dual Spatial Filtration)

Evaluation of residual stress/strain
in a silicon semiconductor ………………………………… 8
Wavenumber reproducibility in the Si peakStress/strain distribution near interface between nitride ﬁlm
and Si substrate(Tips) Simultaneous measurement with a Ne lamp

Measurement of physical properties of a semiconductormaterial using photoluminescence ……………………… 10
PL measurement of InGaN
Combined PL/Raman analysis of SiC
Combined PL/Raman analysis of MoS2

Evaluation of the anode

Evaluation of multi-layer ceramic capacitors …… 15
Structural phase transition of BaTiO3 powder by heating/cooling(Tips) Heating/cooling stage
Evaluating the deterioration of an MLCC using an
acceleration test

Evaluation of carbon materials ………………………… 17
Raman imaging of graphene
Evaluation of carbon nanotubes in the low wavenumber
regionExcitation wavelength dependence of carbon nanotubes
Analysis of DLC coating distribution of a blade edge
Raman measurement of DLC coating on plastic bottles(Tips) Laser adjustment and optimization(Tips) Automatic grating switching mechanism

Evaluation of inorganic materials ……………………… 21
Detection of diﬀerences in crystal structure in inorganic
materials
Analysis of surface iron oxidation states(Tips) SSI (Surface Scan Imaging)2

Polymer
Evaluation of the physical propertiesof polymers………………………………………………… 24
Visualization of sea-island structure of a polymer blend
Evaluation of crystallization in a micro area on a plastic
bottleNon-destructive depth proﬁle measurement and MCR
analysis of polymer laminate
Evaluation of the orientation of polyethylene by Raman
spectroscopy

Biotechnology
Analysis of the orientation and secondary
structure of spider silk………………………………………… 27(Tips) Polarization measurement using IQ Frame

Environmental science
Analysis of microplastics …………………………………… 30
Analysis of AMPs

Evaluating the crystal polymorphs in pharmaceuticaldevelopment………………………………………………… 37
Measurement of trehalose polymorph
Measurement of indomethacin polymorph
Measurement of carbamazepine polymorph
Temperature stability evaluation of pharmaceutical drug
(Tips) Low wavenumber measurement

Art scienceNon-destructive analysis of artworks andother cultural assets …………………………………………… 40
Qualitative analysis of colorants
Combined analysis of toner pigment
Non-destructive analysis of archaeological art

Forensic science
Forensic analysis of vermillion ink …………………… 43

Combined IR/Raman analysis of plastic particles

Measurement using 532 nm laser(Tips) Combined analysis by IQ Frame

Measurement using 785 nm laser(Tips) JASCO Particle Analysis

Analysis of the results

Pharmaceutical
Evaluating the distribution of components inpharmaceutical tablets ……………………………………… 35
Component distribution in a pharmaceutical tablet

Commercial pen ink identiﬁcation ina handwriting document …………………………………… 45
Raman spectra of each pen
Determining the stroke order of intersections
using Raman spectroscopy(Tips) Spectral search program - KnowItAll -(Tips) Analysis Wizard

3

RAMAN SPECTROSCOPY APPLICATIONS - SEMICONDUCTOR -Evaluation of SiC semiconductors
Recently, silicon carbide (SiC) has been attracting attention in thefield of power devices which can convert and control electric power.
Compared with silicon (Si), it has good performance including low
on-resistance, high-speed switching and good thermal stability.
Since it can be used in the manufacture of miniaturized powerdevices with low power consumption, it is expected to provide ahuge impact on energy saving and CO2 emissions. In addition, since
it has the superior features of high breakdown voltage and hightemperature operation, it can be used under severe environmental
conditions that can be challenging for Si based semiconductors.

Wafers with dimensions up to 8 inch can be measured using alarge sample compartment making it possible to evaluate full-sized samples without damage (Fig. 1).
This section shows the evaluation examples of SiC by Raman
spectroscopy.

1) NEDO, TSC foresight vol. 103, https://www.nedo.go.jp/
content/100939129.pdf (2021).

SiC semiconductors can be used where high voltage andtemperature are considered important in a variety of fields thatneed high electric power (such as power generation, electricvehicles, railways) from the aspect of CO2 emission reduction1).
SiC semiconductors are a key factor in achieving carbon neutrality;
This has led to an increase in R&D of high quality semiconductormaterials.
Raman spectroscopy is often used for investigating fundamental
chemical composition, orientation, crystallization, density, stressand temperature of semiconductor materials, it can also be usedfor various structure-property evaluations such as impurities,
defects in the semiconductor materials and composition ratio of
mixed crystal semiconductors. Raman spectroscopy has also beenutilized for estimating carrier density of group III-V semiconductors,
which Hall effect measurement with a formed electrode is applied
for in general.

4Fig. 1 Large sample compartment for 4/6/8 inch wafer.

Evaluation of polymorphism in SiC
It is well known that SiC crystal structure has more than 200
different polymorphs depending on the atomic arrangement;each of these has different physical properties. Among thesemany polymorphs, the most valuable start from 4H, which has
the largest band gap and high mobility. One current hot topic
and a significant challenge has been to selectively grow thesecrystals. Raman spectroscopy is a proven tool used to evaluate
and determine the type of polymorph by analysis of peak
patterns due to lattice vibrations in the crystal structure; theseappear in the low wavenumber region where measurement ismore difficult when using IR spectroscopy.

Fig. 4 Raman imaging measurement result.
(green: TO peak intensity of 3C-SiC, blue: FTO peak intensity of 4H-SiC)

This section shows the results of Raman measurement of two
different SiC monocrystals with 0.33 mm thickness preparedby vacuum sublimation under various conditions (Fig. 2). By
comparing the measured spectra in the range of 150–200
cm-1 and 700–800 cm-1 with reference spectra in a publishedpaper2), the two samples were determined to be 6H and 4H
polymorphs, respectively.
2) Nakashima, S. & Harima, H., Phys . Stat . Sol . (a) 162, 39 (1997).

Defect (3C-SiC)

Int.

(Raman application data: 220-AN-0008)
We would like to express our sincere gratitude to Prof. Inushima andMr. Ohta of Course of Electrical and Electronic System in Tokai
University, Japan, for providing the samples in this evaluation.
A1 (FLO)

Substrate (4H-SiC)
1300

E2 (FTO)1000

E2 (FTA)

Furthermore, Raman spectroscopy can provide depth profile
information in addition to surface analysis.

Int.

As an example, a depth profile measurement of a 3C-SiC defectwas performed (Fig. 6). 3C-SiC was distributed in the depth
direction as well as at the surface.

E2 (FTO)

4H-SiC

Surface

E2 (FTA)
1100 1000

800

100

Fig. 5 Raman spectra of each part.

6H-SiCA1 (FLO)

500-1

Raman Shift [cm ]

600

400

200

100

0

-1

Raman Shift [cm ]Fig. 2 Raman spectra of 6H-SiC and 4H-SiC polymorphs(532 nm excitation)

Distribution analysis of 3C-SiC on 4H-SiC wafer
Raman imaging measurement can detect the location ofpolymorphs, which differ from the wafer substrate. In orderto verify its usability, Raman imaging measurement of 3C-SiC
defects in a 4H-SiC wafer was performed (Figs. 3, 4 and 5).
Distribution information of the defects could be obtained clearlyby creating a chemical image of the specified bands.

1

Z [µm]

2
3
4
5

Inner
Fig. 6 Stack view of defect part (3C-SiC).

Approx. 150 µm

Evaluating carrier density in SiC
In the manufacturing process of semiconductor materials it is
not unusual to dope the pure semiconductor with an impurity toincrease the density of free electrons and electron holes as thecarrier. Raman spectroscopy is a simple method to estimate thecarrier density in n-type semiconductors.

Fig. 3 Observation views of 4H-SiC wafer sample and defect in the wafer.

5

In polar semiconductors (such as GaN, GaAs and SiC),
longitudinal waves and transverse waves due to lattice vibrationin the crystal can be observed separately in a Raman spectrum.
Collective oscillation of free electrons that exist in n-typesemiconductors as the carrier is called a plasmon, which is alongitudinal wave similar to a sound wave. This oscillation is
the same type of wave as the longitudinal wave due to lattice
vibration, longitudinal optical (LO) phonon and therefore theyinteract with each other. This interaction becomes greater withincreasing carrier density and this relevancy makes the peak
position of the longitudinal wave due to lattice vibration shift toa higher wavenumber. Such peak shifts are named as L+ and
L–. In the evaluation of SiC, only the L+ peak can be observed
using Raman spectroscopy. It is well known that this peak shifts
to higher wavenumbers with broadening peak shape when the
carrier density increases, and accordingly, the absolute carrier
density can be evaluated from the peak position.

Non-destructive depth profile measurement of
4H-SiC epitaxial layer
Raman spectroscopy can provide depth profile information
without destroying the sample.
In this example, a depth profile measurement of a 4H-SiC
wafer was performed (Fig. 8). 30 spectra were obtained at 0.5µm intervals from a focal point at the surface on the wafer. It
was confirmed that the longitudinal optical (LO) peak of 4H-SiC(950 to 1000 cm-1) changed depending on the depth. This
demonstrates that the epitaxial layer with low carrier density
was on the surface and the high carrier density was in the inner
layers. It should be noted that the epitaxial layers with high/low
carrier density could be clearly observed using JASCO's uniqueconfocal optical system - Dual Spatial Filtration (DSF: refer topage 7). It is considered that the Z distance (moving distanceof the stage) multiplied by the refractive index of the SiC is theactual depth.

This section shows the results of Raman measurement of 3
types of 4H-SiC monocrystal grown to 0.33 mm thickness using
several different conditioned sublimation methods (Fig. 7).
As the carrier density increased, the peak shape became
broader and the peak shifted to higher wavenumbers.

Depth profile measurement(Raman application data: 220-AN-0008)

Low carrier density:
4H-SiC epitaxial layer(thickness: 12 µm)

We would like to express our sincere gratitude to Prof. Inushima andMr. Ohta of Course of Electrical and Electronic System in Tokai
University, Japan, for providing the samples in this evaluation.

TO

High carrier density:
4H-SiC epitaxial layer

4H-SiC wafer structure diagram

Carrier density

LO

Low

LO

Low carrier density
Surface

Int.

High
1100

1000

800

600

400

200

Int.

100

-1

Raman Shift [cm ]InnerHigh carrier density

several 1016 cm-3
several 1017 cm-3

1100

Low

several 1018 cm-3

100

Fig. 8 Non-destructive depth profile measurement of 4H-SiC wafer.

High

1000

950

900

-1

Raman Shift [cm ]Fig. 7 Change of Raman spectrum of 4H-SiC due to carrier density.
(532 nm excitation)

6

200

-1

Int.

1050

400

600
Raman Shift [cm ]Carrier density

1100

800

Evaluation of component in a multi-layer structureof SiC wafer using DSF and MCR analysis
It is often difficult to perform depth profile measurementof samples with multiple or thin epitaxial layers by Raman
spectroscopy because the spectra may become mixed. Dual
Spatial Filtration (DSF) offers significantly higher spatial resolution
measurement. Also, multivariate curve resolution (MCR) analysis
can easily deconvolve each component spectrum, so that thecarrier densities of each layer can be evaluated (Fig. 9).

Tips
DSF
(Dual Spatial Filtration)
DSF is the mechanism that consists of the confocal
aperture and pinhole slit, and enables to improve thedepth resolution (1 µm at Si substrate). Improvementof depth resolution makes it possible to obtain the
Raman scattering light of the measurement point(focal plane) only (Fig. 10).

Depth profile measurement<Slit confocal optics>
Epitaxial layers

Raman scattering lightby laser irradiation

Substrate

Raman scattering lightfrom measurement point

SiC wafer

SampleSurfaceInt.

Objective
lens

Slit

<DSF confocal optics>
Raman scattering lightby laser irradiation

Raman scattering lightfrom measurement point

Inner
1100

1050

1000

950

900

Raman Shift [cm-1]

MCR analysis
SampleObjective
lens

PinholePinholeFig. 10 Principle of DSF.

Surface layer

Int.

Middle layer

Substrate
1100

1050

1000

950

900

Raman Shift [cm-1]

Fig. 9 Non-destructive depth profile measurement of SiC wafer.

The Intensity profile of a spectrum can provide visual information
about epitaxial layer location3). In addition, since the carrier
density quantified by secondary ion mass spectrometry (SIMS)
measurement is correlated with longitudinal optical (LO) peakshift, it is possible to estimate the carrier densities of each layerusing principal component spectra3).
3) Tawara, T., Ryo, M. & Miyazato, M., Fuji Denki Giho vol. 90
no.4, 214-218 (2017).

7

RAMAN SPECTROSCOPY APPLICATIONS - SEMICONDUCTOR -Evaluation of residual stress/strain
in a silicon semiconductor
As the dimensions of semiconductor devices continue to shrink,there is an increased risk of cracking due to extremely localized
stress and strain. It is therefore important to have an effectivemeans of evaluating the stress/strain in this type of material. Raman
spectroscopy is a powerful tool for investigating the distribution ofstress/strain at high spatial resolution in different regions of a target
sample.
Peak shift in a Raman spectrum is a useful reference for determining
the extent of stress/strain in a crystalline material. Applied stressdecreases the lattice constant, with the peak position shifting tohigher wavenumbers. Conversely, strain shifts the peaks to lower
wavenumbers (Fig. 1).
StressStrainFree

Peak shift521This section shows the evaluation of residual stress/strain in a Si
semiconductor by Raman spectroscopy.
1) Anastassakis, E., Pinczuk, A., Burstein E., Pollak, F. H. &Cardona M., Solid State Commun. 8, 133 (1970).

Wavenumber reproducibility in the Si peak
Since the shifts in silicon peak position are sensitive to changes
in environmental temperature and overall optical system rigidity,
the stability of the entire Raman microscope is important forevaluating very slight residual stress/strain. In order to verify thestability of the Raman microscope, a time-course measurementof the silicon peak was performed (Fig. 2). Peak shift of 0.01 cm-1
or less could be detected, and this result demonstrates that thissystem has high stability.
521.0

Strain520.9

Peak shift

520.5

519
-1

Raman Shift [cm ]250 MPa per 1 cm-1 peak shift (Si)

Fig. 1 Stress/strain evaluation of Si.

Peak position of Si [cm-1]

StressIn the case of crystalline silicon (Si), a peak shift of 1 cm-1 corresponds
to a pressure of 250 MPa 1), and the stress/strain can be evaluatedby comparison with free crystalline Si as a reference.

520.8
520.7

Standard deviation: 0.0059 cm-1

520.6
520.5
520.4
520.3
520.2
520.1
520.0

0

500

1000

1500

2000

2500

3000

Time [sec.]

8

Fig. 2 Si peak position monitoring result.

3500

Stress/strain distribution near interface between
nitride film and Si substrate
Since Raman spectroscopy can be used to evaluate thestress/strain in a Si substrate, it is also possible to observe the
distribution of stress/strain using Raman imaging measurement.
An approximately 60 µm square region at the interface between
a Si substrate and a nitride film was evaluated using Raman
imaging measurement to determine the stress/strain distribution
within the substrate (Figs. 3, 4 and 5). Chemical imaging of theSi-peak position revealed that the stress/strain was generated
in the nitride film and Si, respectively. These results indicatethat Raman microscopy is an effective tool for evaluating the
distribution of stress/strain at micron scale.

Tips
Simultaneous measurement with a Ne lamp
NRS series Raman microscopes include a Ne lamp
(as standard) for wavenumber correction. Monitoringof the Ne peak position allows discrimination of shift
caused by temperature and the stress/strain in thesample. By measuring the sample Raman spectrum
and emission lines of a Ne lamp simultaneously, It is
possible to obtain an extremely accurate peak shift
from the emission line spectrum of the Ne lamp (Fig.
6). In the following cases it is recommended that a
Ne lamp is used for stress/strain measurement. (1) the
measurement time is long (2) the thermal drift in the
spectrograph affects the stress/strain measurement.
Si

Nitride filmNeemission line

Int.

Si Substrate
5 µm

650

600

400

200

130

Raman Shift [cm-1]

Fig. 3 Observation view.

Fig. 6 Simultaneous measurement with the Ne lamp.

+50 MPa(+0.2 cm-1)

StressStressStrain5 µm

Strain-50 MPa-1(-0.2 cm )Fig. 4 Raman imaging measurement result.
(Si peak position)

StressStrain

Int.

540

530

520

510

500

-1

Raman Shift [cm ]Fig. 5 Raman spectra of each point.

9

RAMAN SPECTROSCOPY APPLICATIONS - SEMICONDUCTOR -Measurement of physical properties of asemiconductor material using photoluminescence
Emission intensity and wavelength of photoluminescence (PL) can
provide a variety of information such as the composition ratio andcarrier density in a compound semiconductor. Raman microscopy
can perform PL measurement by changing the optical configuration(laser, filter and grating), and can also perform PL and Raman
measurement at same measurement position.
This section shows the evaluation of semiconductor materials
using PL measurement.

PL measurement of InGaN

PL measurement can be used to observe the specific peaksof InGaN/GaN (Fig. 1), making it possible to evaluate its lack ofcomposition uniformity.
Since PL measurement can detect the differences in indium
composition, the in-plane distribution of the indium composition
ratio can be obtained using PL imaging. As an example, PL
imaging measurement of InGaN was performed (Fig. 2). The
in-plane distribution of the indium composition ratio could beobserved with high spatial resolution.

The band-gap of Indium gallium nitride (InGaN), which is usedin the manufacture of LEDs depends on the indium ratio.
However, increasing the amount of indium makes it difficult togrow uniform crystals and the composition of the crystal tends
to be irregular.

65
Y [µm]

Emission

PL Intensity [arb. units]

Band-edge
emission

200 200 200

7

4
3
2

InGaN

1

GaN

1

2

3

4
X [µm]

5

6

7

0

0

0

Fig. 2 PL imaging measurement result.
(red: peak intensity at 437 nm, green: peak intensity at 454 nm,
blue: peak intensity at 483 nm)

350

400
Wavelength [nm]450Fig. 1 PL spectrum of InGaN/GaN single quantum well strucuture.

10

Combined PL/Raman analysis of SiC
PL measurement can be used to evaluate the defect, bandgap and carrier doping in silicon carbide (SiC), and Raman
measurement can be used for crystallinity and stress/strain.
As an example, PL/Raman imaging measurements of SiC were
performed at the same position (Figs. 3, 4 and 5). Emission
from defects and impurities could be observed with PL imaging
measurement (blue), and the location of different crystal
structures could be observed by Raman imaging measurement(red). Combined analysis by PL/ Raman measurement provides
comprehensive and complementary information.

First, PL imaging of MoS2 was performed (Figs. 6 and 7).
Although no remarkable change could not be found with visible
observation, the modulation of band-gap by electron beam
exposure could be confirmed from the PL measurements of theareas exposed and unexposed to the radiation.

Unexposed area

Exposed area
5 µm

Fig. 6 Observation view.
1.86 eV

Unexposured
area
5 µm

Fig. 3 Observation view.

Exposedarea
5 µm

1.79 eVFig. 7 PL imaging measurement result.
(PL peak shift at 670 nm, 532 nm excitation)
5 µm

Fig. 4 PL imaging measurement result.
(405 nm excitation)

Next, Raman imaging measurement of MoS2 was performed(Fig. 8). A Raman peak shift from stress (E12g) could be confirmed
from the Raman measurements of the electron beam exposed/
unexposed areas. Raman measurement can also obtain
information about the number of layers and stress/strain bymonitoring the peak shift. Combined analysis with PL/Raman
provides comprehensive evaluation of MoS2.
We would like to express our sincere gratitude to Prof. Nobuyuki
Aoki of Chiba University, Japan, for providing the samples in thisevaluation.
386.1 cm-1

Unexposed area
5 µm

Fig. 5 Raman imaging measurement result.
(532 nm excitation)

Combined PL/Raman analysis of MoS2
Molybdenum disulfide (MoS2) has been attracting attention asa 2D material like graphene and is expected to be the materialof choice for next-generation transistors. PL measurement can
provide band-gap information, which defines the performanceof a transistor; this section shows the result of an MoS2 sample,
when the lower half was irradiated with an electron beam.

Exposed area
5 µm

382.3 cm-1

Fig. 8 Raman imaging measurement result.
(385 cm-1 peak shift; E12g, 532 nm excitation)

11

Tips

Tips

Laser adjustment and optimization

Automatic grating switching mechanism

A focusing optical system (beam expander) suitablefor each laser is very useful mechanism for the user.
This system is adjusted beforehand so that the
optimized light beam is introduced to the objective
lens and the high spatial resolution measurement by
the ideal focusing is performed. In addition, since thelaser switching is performed electrically, it is possible
to perform Raman measurement with high reliability
without frequent optical adjustment.

Gratings can be switched automatically throughsoftware operation, and it is possible to select thegrating suitable for the laser without frequent opticaladjustment. By using different gratings, it is possible
to acquire Raman spectrum in wide wavenumber
region at once, and to acquire one with fine and detaileven in narrow wavenumber region with high spectral
resolution.
G bandRBM

2D bandLaser 1Int.

Laser 2
4000

3000

2000

1000

100

Raman Shift [cm-1]

Laser 3
Beam expander
for each laser

Int.

It is possible to narrow down to theideal spot diameter and obtain highspatial resolution by shaping thebeam to the optimum beam diameter
according to the objective lens.

θ
Φ

300

Φ = 1.22λ/N.A.
N.A. = n sinθ
λ: Wavelength, n: Refractive index
Φ: Spot diameter of focusing point

200

100

Raman Shift [cm-1]

Measurements of carbon nanotube using different gratings(green: 300 L/mm, blue: 600 L/mm,
red: 1200 L/mm, pink: 2400 L/mm)*RBM (Radial Breathing Mode) : Refer to page 18.

12

RAMAN SPECTROSCOPY APPLICATIONS - MATERIAL SCIENCE -Evaluating materials in rechargeable batteries
Rechargeable batteries are widely used in daily life. The mostcommon rechargeable battery is the lithium-ion battery, which has
become widespread as the power supply for mobile devices. It
consists of many materials (such as metal oxides, carbon, polymers
and organic solvents) and the qualities of these components affectthe overall battery performance (Fig. 1).
Electrolyte
Separator
CathodeAnode
Copper current
collector

Lithium transition metal
complex oxide

Raman spectroscopy is a powerful tool to evaluate the material
quality in rechargeable batteries. It can be used for surface
analysis of the anode and cathode, and for deterioration analysis
of electrodes by monitoring the differences in crystal structure.
This section shows an example of the analysis of materials foundin a rechargeable battery by Raman spectroscopy.

Evaluation of the cathode
Transition metal complex oxides (such as LiCoO2) are used as
the cathode of lithium-ion batteries. Raman spectroscopy canbe used to measure the spectrum of LiCoO2 (Fig. 2) and canbe used for deterioration analysis of the product by monitoring
the changes.
LiCoO2

Electron
Anode

CathodeAluminium current
collector

Lithium ion

CarbonInt.

Fig. 1 Lithium ion battery diagram (charge).

In the future, larger batteries will be required for a variety of uses,
such as automotive, consumer electronics, energy storage systems,
industrial etc., requiring high reliability as well as high capacity andhigh durability. Therefore, manufacturers are improving quality inexisting lithium-ion technology and developing next-generation
rechargeable batteries whose performance exceeds the batteries
currently being used.

2000

1000

100

Raman Shift [cm-1]

Fig. 2 Raman spectrum of transition metal complex oxide (LiCoO2).

13

In addition, information about the size and distribution ofactive materials can be obtained using Raman imaging1), 2).
Raman spectroscopy is also useful for analyzing the battery
electrolyte1), 2).

Evaluation of the anode
Carbon is mainly used for the anode in lithium-ion batteries.
Since Raman spectroscopy can detect structural changes incarbon with great sensitively it is typically used to perform thismaterial evaluation. Information about quality of graphite can beobtained from the full width at half maximum (FWHM) of the Gband (around 1550 cm-1) and the intensity ratio between the Dband (around 1350 cm-1) and G band (Fig. 5).

1) Celik-Kucuk, A., Yamanaka, T. & Abe T., Solid State Ionics,
357, 115499 (2020).
2) Yamanaka T. et al, ECS Meeting Abstracts, MA2020-01,
203, (2020).

Tips

G bandAtmospheric shielding cell andelectrochemistry cell

D bandRaman spectroscopy is non- contact, non-destructive
and can be used to perform analysis that is difficult forother techniques (such as in-situ measurement).
An atmospheric shielding cell can be loaded in a
glove-box with an inert gas to seal the sample in ananaerobic environment (Fig. 3). This makes it possible
to evaluate sensitive materials such as the electrodematerial without deterioration or oxidation.

After deterioration
Int.

G bandBefore deterioration
2000

1500

1000

Raman Shift [cm-1]

An electrochemistry cell can be used for monitoring
the behavior of an electrode interface during charge/
discharge cycles.

Fig. 5 Raman spectra of graphite.

Raman imaging can check the quality distribution of the carbon
material. In this example, Raman imaging of a graphite sample
was performed (Figs. 6 and 7). Checking the peak intensity
ratio between D and G bands and the FWHM of G band, thedistribution of crystallinity and material quality on the electrodesurface could be visualized.
0.53

Fig. 3 Atmospheric shielding cell.

Evaluation of the binder
PVDF is a material commonly used as the binder in lithiumion batteries, and its spectrum can be obtained by Raman
spectroscopy (Fig. 4). In addition, distribution information of theactive material and binder can be obtained by Raman imaging
and is useful for evaluating the lifetime and performance oflithium-ion batteries.

10 µm
0.33

Fig. 6 Raman imaging measurement result.
(peak height ratio between D band and G band)
PVDF

31

Int.

3600

3000

2000

1000
-1

Raman Shift [cm ]Fig. 4 Raman spectrum of PVDF.

14

150

10 µm

23

Fig. 7 Raman imaging measurement result.
(FWHM of G band)

RAMAN SPECTROSCOPY APPLICATIONS - MATERIAL SCIENCE -Evaluation of multi-layer ceramic capacitors
Recently, the demands for multi-layer ceramic capacitors (MLCC)
have been increasing largely because of the increased use ofelectronics in automobiles and the implementation of autonomous
driving systems as well as the progress of technologies such as
5G, IoT and artificial intelligence (AI).
MLCCs have the structure where the ceramic dielectric andelectrodes are stacked alternately, with terminals at the ends,
which are used for storing electrical energy (Fig. 1).

Terminal

Metallic
electrodes

This section shows the evaluation of structural change of BaTiO3,
and also shows an example of BaTiO3 in an MLCC.

Structural phase transition of BaTiO3 powder byheating/coolingTemperature-dependent Raman measurements were made of
BaTiO3 powder (reagent) using a heating/cooling stage (-190 °C
to 190 °C) (Fig. 2). The spectral shape changed with temperature,and it was confirmed that the phase transition through 4 different
BaTiO3 polymorph occurred. The tetragonal structure which isstable at room temperature (RT) is ferroelectric, and is changed
to cubic structure, which is paraelectric at temperatures higher
than 100 °C. Raman microscopy with a heating/cooling stage
can be used to sensitively evaluate the thermal changes incrystal structure.

Ceramic
dielectric

Cubic
Tetragonal(RT)
190 °C
140 °C
90 °C

Fig. 1 Multi-layer ceramic capacitor (MLCC) diagram.

Barium titanate (BaTiO3) is widely used as a ferroelectric material in
MLCCs. It is well-known that the phase transition of BaTiO3 crystal
structure changes with temperature, causing the permittivity to drop
dramatically at the Curie temperature. Therefore, it is important toevaluate the quality of BaTiO3 to improve its performance and thereliability of MLCCs.
Raman spectroscopy is well suited for the quality evaluation of
BaTiO3. It can be used to monitor the changes in crystal structure,
because Raman spectra are extremely sensitive to the differencesin crystal structure.

Int.

Orthorhombic

30 °C
0 °C
-20 °C-40 °C-100 °C

Rhombohedral-150 °C-190 °C
1000

800

600

400

200

100

-1

Raman Shift [cm ]Fig. 2 Raman spectra of heated/cooled BaTiO3.

15

A heated MLCC cross-section was evaluated using Raman
imaging (Figs. 3 and 4). From the results, it is assumed that there
were localities of structural change at each point in capacitor.

BaTiO3 layer

Evaluating the deterioration of an MLCC using an
acceleration test
This section shows the results of deterioration in an MLCC using
an acceleration test (Figs. 5 and 6). Raman spectra provide
information for evaluating the detailed status (such as crystal
structure) of BaTiO3 layers in MLCC. In addition, Raman imaging
provides information about the distribution of deterioration.

Ni layer
5 µm

Fig. 3 Observation view.

10 µm

A

5 µm

30 °C

5 µm

150 °CB

Fig. 4 Raman imaging measurement results at each temperature.
(peak area at 520 cm-1)

Tips

Fig. 5 Observation view and Raman imaging measurement result.
(FWHM of 520 cm-1)

Heating/cooling stage
The optional stage for heating/cooling the sample,
and depending on the required temperature range,
the user can select one of several types. In addition,
the dedicated software that controls the temperatureprovides automated measurement to start from a set
temperature, and can also set a heating/cooling ramp
and synchronize measurement.

(A)

Int.

(B)

1000

800

600

400

Raman Shift [cm-1]

Fig. 6 Raman spectra at each point.

Heating/cooling stage

16

200

100

RAMAN SPECTROSCOPY APPLICATIONS - MATERIAL SCIENCE -Evaluation of carbon materials
Materials that consist of carbon have various physical properties
depending on the bonding between the atoms and their spatial
arrangement, which are collectively known as "carbon materials".
Utilizing their diverse features, carbon materials are now widelyused in different fields. Raman spectroscopy is one of the few
methods that can be used to evaluate the physico-chemical
properties of carbon materials. Since Raman spectroscopy canmeasure characteristic spectra of each carbon structure, it can beused to elucidate many of these qualities (Fig. 1).
This section shows examples of carbon materials analyzed using
Raman spectroscopy.

DLC film

Graphene is a single-layer carbon molecule that has ahexagonal honeycomb structure. In comparison with othercarbon materials, graphene is a conductor with the following
properties: thin, mechanically strong, transparent and flexible.
Single-layer graphene, like multi-layer graphene, has a varietyof features that have proven to be useful in many applications.
Since the physical (electrical) properties of graphene differ
depending on its layer structure, it is important to be able toevaluate this. In addition, during the manufacture of graphene
structural defects can occur; a simple and rapid quality control
method is required.
Raman spectroscopy can detect the layer structure, defects
and edge structure of graphene with great sensitivity. It is
well-known that peak shape, width and position of the G' (2D)band (around 2700 cm-1) can vary depending on the numberof layers, and it can also be applied to the quality evaluation ofgraphene (Fig. 2). Information about defects and edge structureof graphene can be obtained clearly from the D band (around1350 cm-1) (Fig. 2).

Carbon black

Carbon fiber
Int.

Raman imaging of graphene

Fullerene (C60)

Creating a chemical image of the specified bands provides avisualization of the distribution of the layer structure, defects
and edge structure of graphene (Fig. 3).

Graphite

Carbon nanotube

Diamond

1800

1600

1400

1200

1000

-1

Raman Shift [cm ]Fig. 1 Raman spectra of each carbon material.

17

Evaluation of carbon nanotubes in the low wavenumber region

D bandG' bandG bandRaman spectroscopy can be applied to the analysis of singlewalled carbon nanotube (SWNT). SWNT shows characteristics
typical to metal and semiconductors due to different electronic
structure depending on its chirality and tube radius. Therefore,by exciting the molecules with energy corresponding to theelectronic transition (resonance Raman), information about thechirality and tube radius can be obtained.

Int.

2850

2500

2000

1500

1200

-1

Raman Shift [cm ]This section shows the results of Raman measurement of two
SWNTs with different methods of synthesis (Samples A and B)(Figs. 4 and 5). In the Raman spectra, three specific bands wereobserved. The G band (around 1550 cm-1) corresponds to thevibrational mode assigned to graphite. In metal SWNT, a BWF(Breit-WignerFano) type spectrum is observed in the lower
wavenumber range of the G band. The D band (around 1350cm-1), which is attributed to a defect often used in the evaluationof crystallization. In addition, the band appearing in the low
wavenumber region corresponds to the radial breathing mode(RBM), which correlates to the nanotube stretching diametrically,
and can be used to estimate the diameter of carbon nanotubes.

Fig. 2 Raman spectra at each point (graphene).

(Raman application data: 170-AN-0001)

532 nm excitation (2.33 eV)

G bandD band

Int.

BWF

1800

1600

1400

1200

-1

Raman Shift [cm ]Fig. 4 Resonance Raman spectra of SWNT.
(upper: sample A, lower: sample B)

The number of layers
Armchair edge

Fig. 3 Observation view (left) and Raman imaging measurement result(right) of graphene.
(green: G band, red: D band)

Int.

M1

350

300

S3

200

100

-1

Raman Shift [cm ]Fig. 5 Resonance Raman spectra of SWNT in the low wavenumber region.
(upper: sample A, lower: sample B)(M1: resonance of metallic SWNT, S3: resonance of semiconducing SWNT)

18

Excitation wavelength dependence of cabon
nanotubes

Analysis of DLC coating distribution of a blade edge

In order to verify the dependence on excitation wavelength,
Raman measurements of SWNT (Sample B) were performed
using several different excitation lasers (Figs. 6 and 7). Using
resonance Raman, measurement can be made selectively forspecific chirality and tube radius, depending on the excitation
wavelength. Therefore, the shape of the Raman spectrachanges significantly depending on the selected excitation
wavelength. 532 nm excitation is mainly resonant with metallic
SWNT, 633 nm with both metallic and semiconducting SWNT,
and 785 nm with semiconductor SWNT.
(Raman application data: 170-AN-0001)

Diamond-Like Carbon (DLC) is an amorphous carbon material
that has an intermediate molecular structure between diamondand graphite. DLC coatings are used to improve abrasion
resistance, chemical resistance etc., and is used as a coating
for cutting tools, automotive components, medical equipment,
optical components, and many other products.
It is well-known that Raman spectroscopy can acquire structural
information about carbon materials (such as DLC, diamond andgraphite) with significant sensitivity. The Raman spectrum of DLC
consists of a D band (around 1350 cm-1) and a G band (around1550 cm-1). Generally, curve fitting to decomposition into G and
D bands can be used for evaluating the quality of DLC, such as
crystallinity (Fig. 8).

785 nm excitation (1.58 eV)Int.

Fitted line

G band633 nm excitation (1.96 eV)Int.

532 nm excitation (2.33 eV)

1800

1600

1400

D band1200-1

Raman Shift [cm ]

1800

1600

1400

1200

1000

Raman Shift [cm-1]

Fig. 6 Raman spectra of SWNT (excitation wavelength dependence).

Fig. 8 Decomposition of Raman spectrum (DLC).

As an example, Raman imaging of a DLC-coated blade edge
was performed (Fig. 9). Since the sample had a significant slope,measurement was performed using surface scan imaging (SSI),
which adjusts automatically to the surface topography (refer topage 23). The results confirmed that there was a DLC coatingon the edge of the blade. Creating a chemical image made it
possible to visualize the distribution of DLC the coating.

S2
M1

S2

Int.

M1

M1

S3

Blade edge
350

300

200

100

(DLC)

-1

Raman Shift [cm ]Fig. 7 Raman spectrum of SWNT in low wavenumber region.
(excitation wavelength dependence)(M1: resonance of metallic SWNT, S2, S3: resonance of semiconducing SWNT)8060
Z [µm] 4020
0200

Blade root

250

150

300

200
100

Y [µm]

50

50
0

100

150
X [µm]

0Fig. 9 DLC coating distribution on blade edge.

19

Raman measurement of DLC coating on plastic
bottles
DLC coatings have also been used to improve the gas barrier
properties of plastic bottles. This section reports the analysis of
DLC coated plastic bottles using Raman spectroscopy.
The samples analyzed were two plastic bottles with a visualappearance of slightly different colors (Fig. 10). From this point
forward, samples from the slightly colored plastic bottle andfrom the clear bottle are each referred to as “coated” and "noncoated", respectively.

OutsideDLC

Int.

InsideMeasurements were made of both the interior and exterior
surfaces of each plastic bottle, and the Raman spectra werecompared.

3700

3000

2000

1000

100

Raman Shift [cm-1]

Fig. 12 Measurement of coated sample.

For detailed analysis, a difference spectrum from the coatedsample was calculated from both spectra (Fig. 13). A library
search identified the difference spectrum as DLC. Only PET
was identified on the exterior of the coated sample, but on theinterior surface DLC was found in addition to PET, indicating thatthe inside of the plastic bottle was coated with DLC.

CoatedIn addition to identification, the quality of the DLC coating canalso be evaluated from its spectrum; the D band (sp3 bonds inthe material lattice) and G band (sp2 bonds in the material lattice)
can be deconvolved using curve fitting (Fig. 13). It is possible toevaluate characteristics such as abrasion resistance from thepeak positions, half widths, peak ratio etc.

Non-coatedFig. 10 Measurement sample.

(Raman application data: 020-AN-0023)

In the case of the non-coated bottle, similar polyethylene
terephthalate (PET) spectra were obtained for both interior andexterior surfaces (Fig. 11). For the coated bottle, different spectra
were obtained from the interior and exterior measurements (Fig.
12).

Fitted line

G bandInt.

OutsideD bandInt.
1800

Inside

3700

3000

1400

1200

1000

900

Raman Shift [cm-1]

2000

1000
-1

Raman Shift [cm ]Fig. 11 Measurement of non-coated sample.

20

1600

100

Fig. 13 Difference spectrum of coated sample (Inside – Outside)and D and G bands deconvolution.

RAMAN SPECTROSCOPY APPLICATIONS - MATERIAL SCIENCE -Evaluation of inorganic materials
Several inorganic compounds have the characteristic that their
physical properties differ depending on crystal structure, andtheir application in a variety of fields utilizes these characteristics.
Therefore, understanding the crystal structures of inorganic
materials is important, which can lead to improvements in material
quality.
Since Raman spectroscopy is excellent for measurement in the low
wavenumber region, it can be used to observe the specific lattice
vibrations that are transmitted through an entire inorganic crystal.
This makes Raman spectroscopy a useful tool for evaluating the
crystal structure of inorganic materials. In addition, it can also be
used to clearly detect the differences in crystal structure, because
the lattice vibration varies depending on the arrangement, electriccharge, bond length, and bond strength of atoms.

Anatase
Rutile

Int.

800

400

200

100

200

100

Raman Shift [cm-1]

Fig. 1 Raman spectra of TiO2.

This section shows the evaluation of inorganic materials using
Raman spectroscopy.

Detection of differences in crystal structure
in inorganic materials
Raman measurements of titania (titanium dioxide, TiO2) andzirconia (zirconium dioxide, ZrO2) were performed (Figs. 1 and
2). For both samples, a difference in crystal structure could bediscriminated in the low wavenumber region (less than 800 cm-1).
Since the vibrational frequency of a crystal varies depending on
the arrangement of atoms, even if the chemical formula is same,
Raman spectroscopy can detect the difference in polymorphs.

600

Tetragonal
Monoclinic
Int.

800

600

400
Raman Shift [cm-1]

Fig. 2 Raman spectra of ZrO2.

21

Analysis of surface iron oxidation states
Rust that occurs when ferrous materials corrode has various
oxidation states and crystal structures, such as Fe2O3, Fe3O4,
and FeOOH, resulting in differences in Raman spectra (Fig.
3). This section shows the analysis of oxidation states on thesurfaces of several iron materials using Raman microscopy.

α-Fe2O3 (hematite)

Next, Raman imaging measurement of a sample collectedindoors was performed (Fig. 5). Since the surface of the rusted
metal had a lot of irregularities, surface scan imaging (SSI) wasused for Raman imaging measurement across the irregularities(refer to page 23). In addition, principal component spectra wereobtained using multivariate curve resolution (MCR) analysis.
Using these as reference spectra, a chemical image of thecorrelation (similarity) with the reference spectra was created.
Three different spectra were obtained, and the component
distribution that can be difficult to determine visually was
obtained by creating a chemical image.

Fe3O4 (magnetite)(Raman application data: 170-AN-0021)
α-FeOOH
Int.

β-FeOOH
α-Fe2O3 + Fe3O4

γ-FeOOH
Int.
2500

2000

1000

α-Fe2O3

100

Raman Shift [cm-1]

Carbon + α-Fe2O3 + Fe3O4

Fig. 3 Raman spectra of iron oxides and hydroxides.

First, Raman measurement was made of a sample collected
after environmental exposure (Fig. 4). γ-FeOOH was mostly
detected in this material.

3700

3000

2000

1000

100

Raman Shift [cm-1]

Principal component spectra and library search results

1 1 1

0.7 0.3 0.9

Raman imaging measurement result(Overlaid all in-focus image with 2D display)

γ-FeOOH

15
Int.
10
5

3700

3000

2000

1000

Z [µm]

0100-1

Raman Shift [cm ]-520Fig. 4 Measurement result of the sample collected outdoors.

10
-20

0
-10
0
X [µm]

Y [µm]-10
10

20 -20Raman imaging measurement result(Overlaid all in-focus image with 3D display)
Fig. 5 Measurement result of the sample collected indoors.

22

Tips
SSI (Surface Scan Imaging)
A Raman microscope typically has a confocal opticalsystem, which can obtain Raman spectra with highspatial resolution (submicron order). Therefore, it canbe applied to the characterization of minute foreignmaterial (down to 1 µm) and extremely thin layers.
However, due to the high spatial resolution of theRaman microscope, slight focus deviation on themeasurement surface greatly affects the intensity ofthe spectrum. As a result, Raman imaging of a sample
with an uneven or sloping surface may not provide
results.

In order to solve this problem, JASCO developed
surface scan imaging (SSI). SSI acquires the surface
shape information of the sample as a data matrixof the corresponding height of the stage prior tomeasurement (Fig. 6). Measurement is made while thefocal point is controlled by the stage height following
the surface shape, not by autofocus. By using SSI,
samples with uneven surfaces, which have previously
been difficult to measure, can now be imaged using
Raman micoscopy with high S/N.

180

Z = 250 µm

100
0
780
600

Z [µm]

400
X [µm]
200
0 580

Omnifocal
image

Stage
driving

200
300
400 Y [µm]
500

100

0

3D image200
100

Z [µm]

0
0
780

600

200
400

X [µm]

200

400

Y [µm]

0 580

Z = 0 µm

2D image

Fig. 6 Imaging of an uneven surface using omnifocus (SSI).

23

RAMAN SPECTROSCOPY APPLICATIONS - POLYMER -Evaluation of the physical properties of polymers
Products manufactured from polymers rarely consist of a single
component; combining different polymers can produce materials
with superior properties. Therefore, to understand the qualities ofpolymer materials, it is important to obtain information that simple
visual observation cannot provide.
Raman spectroscopy is used to identify the components in amaterial without destroying the sample and can also determine
information about crystallinity. In addition, the physical propertiesof blended polymers depend on the mixture of ingredients. Raman
imaging provides information about component distribution, whichcannot be obtained by visual observation. Raman spectroscopy
can also be used to analyze the layers in polymer laminates.
This section shows the evaluation of polymer materials using
Raman spectroscopy.

Visualization of sea-island structure of a polymer
blend

5 µm

5 µm

Fig. 1 Observation view (left) and Raman imaging measurement result (right).
(blue: butadiene, green: acrylonitrile styrene)

Evaluation of crystallization in a micro area on aplastic bottle

Blending several polymers can create functional resin materials
with superior physical properties. It is known that the seaisland structure is formed depending on the affinity between
the polymers, and its distribution depends on their mechanicalproperties.

The Raman spectrum of polyethylene terephthalate (PET) (Fig.
2), measured with a full width at half maximum (FWHM) of the
carbonyl group (1730 cm-1) correlates well with the crystallization
density1). This section shows the evaluation of the distribution of
crystallization in a cross-section of a plastic (PET) bottle.

Raman imaging measurement of the surface of a commerciallyavailable Acrylonitrile Butadiene Styrene (ABS) resin (20 µm
× 20 µm region) was performed, and the distribution of two
principal components (butadiene and acrylonitrile styrene) was
visualized using multivariate curve resolution (MCR) analysis (Fig.
1). Raman imaging measurement can visualize the composition
distribution, which cannot be seen in the observation view.

Two samples of a bottle were made, then a cross-section ofeach was prepared using a slicer (location A and B) (Fig. 3),
since Raman imaging of a micro area requires a smooth surface,an HW-1 variable angle slicer (JASCO Engineering Co., Ltd.) wasused to make good cross-section samples.

24

Non-destructive depth profile measurement andMCR analysis of polymer laminate
Stacking several polymers with different properties can produce
a polymer laminate with valuable functionality such as heat
resistance, heat sealability, high rigidity, flexibility, cold resistanceand easy-peeling. Since the component distribution in eachlayer affects the overall functionality of polymer laminates, it isimportant to confirm that the material can be manufactured tothe intended design.

1730 cm-1

Int.

2000

1800

1600

1400

1200

Raman spectroscopy can perform non-destructive depth profile
measurement without having to make a cross-section of thesample. In addition, utilizing multivariate curve resolution (MCR)
analysis, the components in each layer can be easily identified,
and their distribution can then be visualized.

1100

Raman Shift [cm-1]

Fig. 2 Raman spectrum of PET.

In order to verify the structure, non-destructive depth profile
measurement of a polymer laminate was performed (Figs. 5
and 6). Different layers were clearly identified because of the
improved spatial resolution offered by JASCO's unique confocal
optical system - Dual Spatial Filtration (DSF) (refer to page 7).
MCR analysis confirmed that each observed layer agreed very
well with the actual product design.

OutsideInsideB
OutsideA

Inside

1st layer (28.8 µm)
2nd layer (3.0 µm)
3rd layer (23.7 µm)
4th layer (2.4 µm)
5th layer (23.4 µm)
6th layer (44.2 µm)
7th layer (30.0 µm)

Fig. 3 Sampling part and its cross-section.

8th layer (30.9 µm)

Multi-point measurement was carried out at 50 µm steps throughthe sample section, and the FWHM of the carbonyl group was
calculated at each depth (Fig. 4). The FWHM and crystallization Fig. 5 Sample overview (cross-section, left) and component distribution (right).
(red: polypropylene, green: polyethylene, blue: cellulose,
show a negative correlation: as the amount of crystallizationaqua: polyamide, purple: polyurethane)
increases the FWHM becomes narrower. It was confirmed thatboth crystallization distributions differ from each other.
(Raman application data: 030-AN-0002)
Polypropylene

26

B

FWHM [cm-1]

25

Polyethylene

A
Int.

Cellulose24

Polyamide
23

Polyurethane
3700

22

3000

2000

1000

200

Raman Shift [cm-1]
0

100

200

300

400

Measurement position (depth from outside) [µm]Fig. 6 Five principal component spectra and library search results.
(obtained by MCR analysis)Fig. 4 FWHM calculation results in 1730 cm-1.

1) Ian R. Lewis, Howell G. M. Edwards, Handbook of Raman
Spectroscopy (Marcel Dekker, INC., 2001).

25

Evaluation of the orientation of polyethylene byRaman spectroscopy
Plastic string has the property of easily tearing in its crosssection, but with strength in the longitudinal direction. This isbecause stretching the fibers orients the molecular chains,resulting in increased physical strength in that direction. Thissection reports the orientation analysis of a polyethylene string
using polarization measurement with a Raman microscope and
IQ Frame (refer to page 29).

As a result of obtaining Raman spectra by rotating thepolyethylene string through 360°, changes were observed in
the C-C skeletal vibration (Fig. 8). The C-C skeletal vibration isclosely related to the degree of orientation. By plotting the peakintensities at each angle, it was confirmed that a figure-8 shaped
intensity distribution with maxima at 0° and 180° were observed(Fig. 7). Therefore, it is suggested that this polyethylene string
sample was strongly stretched uniaxially in the laser polarization
direction (horizontal direction on the page) at 0° and 180°.

A polyethylene string was fixed to a metal plate and set in the IQ
Frame. For polarization measurement, the analyzer (polarizer)
was placed so that the sample was parallel to the polarization of
the incident light (laser), and Raman scatter polarized parallel tothe incident light was detected. The sample was rotated in 10°
steps using the scale on the IQ Frame, and Raman spectra wereacquired at each of the 36 points (total 360°) using the rotationalalignment function of the IQ Frame. It was confirmed from theobserved image that the same point could be measured ateach angle (Fig. 7).

(Raman application data: 030-AN-0024)

C-C skeletal vibration

0°

Int.

0°

90°

Polarization directionof laser

30°

180°
60°
3800

320

330

340

10

500

20

400

310

30

50
60

290

200

70

280

100

80

270

0

100

250

110

240

120

230

180°

90°

90

260

220

130
210

200

190

180

170

160

150

140

120°

150°

Measurement
point

Fig. 7 Peak intensities plot and observation view.

26

2000

1000

Fig. 8 Raman spectra at 0º, 90º and 180º.

40

300

300

3000

Raman Shift [cm-1]

0100

RAMAN SPECTROSCOPY APPLICATIONS - BIOTECHNOLOGY -Analysis of the orientation and secondary
structure of spider silk
Spider silk is a remarkable protein material with a tensile strength
greater than that of steel, while at the same time being more ductile
than nylon. Spider silk is attracting attention as a source of next
generation materials. It exhibits partial crystalline properties where
the protein molecules are oriented parallel to the fiber axis during
self-assembly. The aggregation of certain amino acid residues inthe silk dictates the elasticity and strength of the material; as a result
there are various types of spider silk. A typical orb web is composedof three fibers each with different physical properties - dragline,
radial line and spiral line (Fig. 1). This section reports the structural
analysis of spider silk proteins by polarization measurement with aRaman microscope.
Radial line

Sticky
ball

0°

90°
Measurement
area

5 µm

Radicalline

Spiralline

Dragline

Polarization
direction

Observation view (100x)

Spiral line

Fig. 2 Definition of polarization direction.
Dragline

Fig. 1 Example of spider silks.

Raman measurements were performed for each of the three typesof spider silks (dragline, radial line, and spiral line) with diametersof several µm, which were sourced from the oriental golden orb
web spider, Nephila clavata. Measurements were performed byrotating the spider silk to align the polarization direction of thelaser light with the axis parallel to, and orthogonal to the fiber axis
(Fig. 2).

First, polarization measurements using a Raman microscopewere performed (Fig. 3). These show the differences in protein
orientation, secondary structure, and amino acid composition. The
amide I and amide III bands are characteristic of protein peptide
bonds, which vibrate in the direction perpendicular and parallel tothe molecular axis, respectively, so that their peak intensity ratios
reflect the magnitude of protein orientation. In addition, it is wellknown that the peak positions of amide I and amide III shift due todifferences in the secondary structure of proteins1).

27

Amide III
Amide I

ProlineCH bending
0°90°

Radial
line

0°
90°

Int.

Spiral
line

0°
90°
1800

1600

1400

1200

1000

Dragline

800

Raman Shift [cm-1]

Fig. 3 Polarization measurements of each spider silk.

Analysis of the peak intensity ratio and peak position of amide I
and amide III show that the dragline and radial line proteins arelargely oriented in the fiber axis direction and contain a large
crystalline component with β sheet structure. This is thought to berelated to the tensile strength of the spider silk in the direction of
the fiber axis.
The spiral line (for catching prey) has low orientation and contains
many α helices and random coil structures. This is thought to berelated to the ductile and elastic properties of the spiral line fiber(Fig. 4). In addition, the spectrum of the spiral line fiber suggeststhat its surface includes proline-rich ‘sticky-ball’ components.
(Raman application data: 210-AN-0019)
This application is the result of a joint research project with Prof. N.
Katayama at the Graduate School of Natural Sciences, Nagoya City
University.

1) Carey, P. R., Biochemical Applications of Raman andResonance Raman Spectroscopies, (Academic Press, New
York, 1982).
β sheetα helix

Random coil

Fiber axis

Dragline
Random coil
α helix
β sheetSticky ballcomponents

Fiber axis

Spiral line

Fig. 4 Image of dragline and spiral line silk fibers.

28

Tips
Polarization measurement using IQ Frame(1) sample rotation

The functions and properties of polymer materials
such as films and chemical fibers are closely related
to the molecular orientation. Non-contact, nondestructive evaluation of the orientation of polymers
can be made to micron resolution using polarization
measurement with Raman microscope.
When performing the polarization measurement byrotating the sample, it is not easy to move the sample
to exactly the same measurement position after
rotating it under the Raman microscope (Fig. 5).
Measurement point
after rotating (laser spot)

Measurement point (laser spot)

Rotates the sample
after measurement.
Rotation

SampleMeasurement point after rotating(2) Stage movement
Moves the stage to measurement point calculated fromthe center coordinates of rotation and the rotation angle.

rotation
Center of rotation

Measurement point
before rotating

Fig. 5 Conventional polarization measurement.

Movem

ent

In order to satisfy the above needs, JASCO hasdeveloped the solution using IQ Frame including
the image matching technique (Fig. 6). IQ Frame
including the image matching technique enables toperform the measurement at the same position evenafter rotating the sample (Fig. 7). This function makes
it possible to perform the polarization measurement
without changing the polarization plane of laser. Since
the polarization plane on the laser beam side is not
moved, a spectrum without the polarization property
influence of optical elements can be obtained. By
continuously rotating the sample, it is possible todraw a profile of peak intensity.

(3) Image matching
Corrects the small displacement of measurement point byusing image matching technique.
Thumbnail image

Fig. 7 Measurement overview (after rotating).

Analyzer
RotationFig. 6 Polarization measurement using IQ Frame.

29

RAMAN SPECTROSCOPY APPLICATIONS - ENVIRONMENTAL SCIENCE -Analysis of microplastics
Microplastics are a major global problem; this form of environmental
pollution is not only found in just about every type of water sourceon the planet, it can also found throughout the food chain withpotential for damage to life on Earth. Recently, the investigation
into microplastics has exploded with scientists around the world
assessing the distribution and effects of these materials witha view to reduce the environmental impact and understand thewidespread implications.

(A)

(B)

Zoom

Zoom

This section shows examples of the evaluation of microplastics
using Raman spectroscopy.

Analysis of AMPs
Airborne microplastics (AMPs, PM2.5) were collected on analuminum oxide membrane filter at the top of Mt. Fuji (in thefree troposphere) at night. These were analyzed using Raman
microscopy (Figs. 1 and 2). Polyethylene terephthalate (PET)and Polyhydroxybutyrate (PHB), a biodegradable plastic wasfound as particulates, with sizes around 5 μm or less. The
results demonstrate that Raman microscopy is well suited for
microplastics analysis.
(Raman application data: 100-AN-0031)

Fig. 1 Observation view.

(A)

PET

(B)

PHB

Int.

We would like to express our sincere gratitude toProfessor Hiroshi Okochi, School of Creative Scienceand Engineering, Waseda University, Japan, forproviding the AMPs samples and for his guidance.
3800

3000

2000
Raman Shift [cm-1]

Fig. 2 Measurement results.

30

1000

100

Combined IR/Raman analysis of plastic particles
Measuring a sample using two complementary analyticalmethods (such as Raman spectroscopy and IR spectroscopy)
can provide more information than one single method, and it
can also increase confidence in the results. When performing
combined analysis, IQ Frame makes sample measurement
using different spectroscopic techniques much more userfriendly (refer to page 33) (Fig. 3). This section shows a sample
evaluation using IQ Frame with IR and Raman microscopes.

First, Raman imaging measurement was performed (Fig. 5).
Principal component spectra were obtained using multivariate
curve resolution (MCR) analysis, and a chemical image wascreated using the specified bands in the principal component
spectra. As a result, carbon was found in addition to four plastics
identified by Raman spectroscopy: polyethylene terephthalate(PET), polyethylene (PE), polypropylene (PP) and polystyrene(PS).
CarbonPS

PP

Raman microscope

30 µm
PET

PE

Raman imaging measurement result(peak heights of the specified bands)

IR microscope

PET (green)

PE (red)Int.

PP (blue)Fig. 3 IQ Frame overview.
(between Raman microscope and IR microscope)

PS (aqua)

A sample sprayed with plastic particles were measured using
Raman and IR microscopy (Fig. 4).

Carbon (white)

3500

2000

300

Raman shift [cm-1]

Principal component spectra
Fig. 5 Measurement result (Raman microscope).

30 µm

Fig. 4 Observation view.

31

Next, utilizing IQ Frame, IR imaging measurement was performed
at exactly the same position measured using the Raman
microscope (Fig. 6). The principal component spectra wereobtained by MCR analysis, and a chemical image was createdusing the principal component spectra. As a result, protein was
found in addition to four plastics: PET, PE, PP and PS.
Protein
PP

PS

Only five components could be detected using either IR or
Raman spectroscopy alone. However, combining analysis fromboth techniques resulted in six different components being
detected.
In order to visualize the complete information, both chemical
images were overlaid (Fig. 7). This showed the protein detectedby IR spectroscopy together with the other five components,
PET, PE, PP, PS and carbon detected by Raman spectroscopy.
These results indicate that using the strong points of both IR/
Raman spectroscopy can be combined to be more useful than asingle analytical technique alone.

Protein

Carbon
PP

PS

30 µm
PET

PE

IR imaging measurement result(score of the principal component spectra)

30 µm
PET
PET (green)PE

Fig. 7 Overlaid chemical image of Raman and IR.

Abs.

PE (red)PP (blue)

PS (aqua)Protein (yellow)

4000

3000

2000

700

Wavenumber [cm-1]

Principal component spectra
Fig. 6 Measurement result (IR microscope).

In the analysis by Raman microscopy, carbon was detectedin addition to the four plastics, whereas in the analysis by
IR microscopy, protein was detected (carbon could not be
detected). These results were obtained because Raman
spectroscopy has low detection sensitivity for protein, but it isalso good for carbon analysis.

32

Tips
Combined analysis by IQ Frame(1) Sample setting

JASCO can provide a microscopic spectroscopic
system for acquiring information at a coordinatedlocation (such as Raman microscope, IR microscopeand UV/Vis microscopic spectrophotometer).

Sets the sample.

Each system can obtain the useful information, andin order to obtain the additional information using
another analytical method, the demands that one
sample wants to be evaluated using several analyticalmethods are increasing. However, it is not easy
to measure the sample at same position between
different microscopes.
In order to satisfy the above needs, JASCO hasdeveloped the solution using IQ Frame including
image matching technique (Fig. 8).

(2) Movement by coordinate information

Moves the stage bycoordinate information
which was obtained inprevious measurement.

Fig. 8 IQ Frame.

The stage is moved to the measurement point roughly
by using the coordinate information, and then is
moved finely by using the coincidence of observation
image (image matching technique) (Fig. 9). Since
IQ Frame can move the stage to the measurement
point with high position accuracy (micron-order), it ispossible to measure the sample at the same position/area easily and rapidly even after changing to theother microscope.

(3) Movement by coincidence of observation image
Corrects the small
displacement of
measurement point byusing image matching
technique.

Fig. 9 Overview of image matching technique.

33

Tips
JASCO Particle Analysis[JASCO Particle Analysis] program is an add-in
package for the [Micro Imaging Analysis] program,and can perform the particle analysis and thestatistical processing. It can automatically binarize
observation images or chemical images, and perform
various particle analyses such as determining
sample size, area, perimeter, horizontal/vertical Feretdiameter, aspect ratio, and circularity. Using thestatistical processing function, a stacked histogramfor each component, a frequency distribution table,a correlation distribution, and a component ratio canbe displayed.

As the measurement/analysis example, Raman
imaging measurements of 2 types of rubber were
performed, and the distributions of additive (calcium
carbonate; CaCO3) in each sample were also
evaluated. Raman imaging measurement could
obtain the distribution information of additive, which
could not be obtained from observation view (Fig. 10).
In addition, the particle analysis of the obtained
chemical image could evaluate the distribution
status statistically (such as size and circularity), anddiscriminate the difference between the samples.
[JASCO Particle Analysis] is effective tool to evaluate
the particles statistically based on the chemical
information of sample.

<EPDM><Natural rubber>

30

30

Average: 4.7 µm

11

10

12

Count

Count

20
14 14

10
5

6

5
2

0

0 1 2 3 4Median: 2.2 µm

20

10

8

3

1

Average: 3.1 µm

24Median: 3.9 µm
20

28 27

3

1

0

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

9

7

5

7
3

Median: 0.72

17

20

19

11
7 7

0.2

0.3

13
9
6
3

1
0.5

0.6

Circularity

0.7

0.8

0.9

1.0

0
0.0

12

11

4

0.4

18

10

8

1 1
0.1

Median: 0.74

6

5 4

0
0.0

23

Average: 0.69

Count

Count

15

9

1

Size [µm]Average: 0.66

101

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Size [µm]

20

2

2

0.1

0.2

4

0.3

5

3 3 3
0.4

0.5

3
0.6

0.7

0.8

Circularity

Fig. 10 Particle analysis of additive (CaCO3) in EPDM and natural rubber.
(upper: Raman imaging measurement results of CaCO3, middle: size histograms, lower: circularity histograms)

34

0.9

1.0

RAMAN SPECTROSCOPY APPLICATIONS - PHARMACEUTICAL -Evaluating the distribution of components inpharmaceutical tablets
The distribution of active pharmaceutical ingredients (APIs) andexcipients in formulated tablet affects its dissolution in the body,and it is important to evaluate this at an early stage in drug
development and quality control.
Raman spectroscopy is well-known as a tool for evaluating theAPIs and excipients in tablets both qualitatively and quantitatively.
Since Raman imaging measurement can visualize the distributionof APIs and excipients, it is a very effective tool in pharmaceuticaldevelopment and quality control.
This section shows the evaluation of component composition in a
pharmaceutical tablet.

Component distribution in a pharmaceutical tablet

50 µm

Fig. 1 Observation view.

Raman imaging measurement was performed on a selectedarea of a tablet (Figs. 1, 2 and 3). Principal component spectra
were calculated by multivariate curve resolution (MCR) analysis.
Creating a chemical image using MCR analysis makes it possible
to visualize the distribution of the APIs and excipients.

50 µm

Fig. 2 Raman imaging measurement result.
(pink: apronalide, aqua: caffeine, blue: lactose,
red: ethenzamide, green: acetaminophen)

35

Tips
Apronalide (pink)

Analysis Wizard
With the [Micro Imaging Analysis] program, it ispossible to create chemical images using peak height,
area, half width, correlation coefficient, multivariate
analysis, etc. with a simple operation.

Caffeine (aqua)Int.

Lactose (blue)

Acetaminophen (green)

Ethenzamide (red)
3500

3000

2000

1000

300

Raman Shift [cm-1]

In addition, color maps can be created by using the[Analysis Wizard] according to the sequence andassist guidance.

Fig. 3 Principal component spectra obtained by MCR analysis.

Int.

3600

3000

2000

1000

100

Raman Shift [cm-1]

Component 1

Int.

Component 2

Component 3
3200

2000

1000
Raman Shift [cm-1]

36

300

RAMAN SPECTROSCOPY APPLICATIONS - PHARMACEUTICAL -Evaluating the crystal polymorphs inpharmaceutical development
Crystal polymorphs are molecules that can exist with the samechemical formula but different crystal structures. Crystal structure
depends on the conditions (such as temperature and pressure)used in the crystal formation process. The resulting crystal
structures are conveniently classified as Forms I, II, III or Ta and Tb.
In medicine, solubility, stability and bioavailability depend on the
crystal polymorphism; compounds with different crystal structures
may even be misidentified as different compounds. Therefore,
research into crystal polymorphism is widely used in areas suchas basic studies, structure/function correlation and industrial
applications including patent registration.
In the pharmaceutical industry, the study of crystal formation
techniques is very important in the control of crystal structure
and the manufacture of high-quality medicines. Although X-ray
diffraction (XRD) and differential scanning calorimetry (DSC) are
widely used in the evaluation of crystal polymorphism, they havesome drawbacks. XRD cannot evaluate thermal stability, and it isdifficult to measure samples with low reflectivity (less diffractionspot) such as amorphous and/or tiny crystals. Conversely, DSC
can be applied to evaluate the thermal stability of a crystal, but
structural information cannot be obtained.
Raman spectroscopy is becoming a commonly used analyticaltechnique for the study of crystal polymorphism, with the following
features.
● Both crystal and amorphous structures can be evaluated.
● Vibrational information of molecules can also be obtained
from high wavenumber spectra.
● High spatial resolution and micro measurement can be
performed.
● Sample preparation is not required.
● It is possible to monitor in-situ structural changes withtemperature.

Presently XRD is used for the basic study of crystal structure forpatent registration, and Raman spectroscopy is also expected
to become a more widely used technique in the pharmaceuticalindustry for patent registration for the above reasons.
This section introduces the evaluation of crystalline polymorphs
using Raman spectroscopy.

Measurement of trehalose polymorph
Raman measurements of trehalose dihydrate and trehaloseanhydrate were performed (Figs. 1 and 2). It was confirmed thatthe peaks of molecular vibration and lattice vibration differs fromeach other due to differences in the crystal structure.

OH (molecular vibration)

Trehalose dihydrate
Int.

Trehalose anhydrate

3550

3400

3200

3000

2900

Raman Shift [cm-1]

Fig. 1 Raman spectra of trehalose.

37

Measurement of carbamazepine polymorph
A commercially available tablet containing carbamazepine
was measured using Raman microscopy (Figs. 4, 5 and 6).
Since Raman spectroscopy can easily detect peaks in thewavenumber region below 100 cm-1, it is possible to identify
differences in the crystal structure. In addition, by creating achemical image, the distribution of the specified crystal structure
can also be visualized (Figs. 5 and 6).

(Lattice vibration region)

Trehalose dihydrate
Int.

Trehalose anhydrate
N
O

200

150

100

50

NH2

Form II

10

Raman Shift [cm-1]

Form IIIInt.

Fig. 2 Raman spectra of trehalose.
(low wavenumber region)

Amorphous

Measurement of indomethacin polymorph
Commercially available indomethacin was measured using
Raman microscopy with and without a low wavenumber
measurement unit (Fig. 3). It is known that indomethacin exists
as three different crystal structures: alpha (acicular crystal), beta
and gamma (plate crystal). Gamma is the most stable structure
followed by the alpha structure. Recently, the alpha polymorph
has been selectively manufactured (and patented) to be used
as a topical medicine for enhanced stability and skin absorption.
The method for effective manufacturing of a specified crystal
structure is extremely important. Since low wavenumber
measurement by Raman spectroscopy provides useful
information about lattice vibration, it can be used to evaluate
crystal polymorphs without any pre-treatment or damage to thesample.

28 cm-1

800

600

400

200

Raman Shift [cm-1]

Fig. 4 Raman spectra of carbamazepine.

1 mm

Cl

O

Fig. 5 Observation view of tablet.

N
O
O
OH

Int.

720

600

400

200

10

Raman Shift [cm-1]

Fig. 3 Raman spectra of indomethacin.
(blue: with low wavenumber measurement unit,
green: without low wavenumber measurement unit)

1 mm

Fig. 6 Raman imaging measurement result.
(Distribution of carbamazepine Form III)38

10

Temperature stability evaluationof pharmaceutical drug
The stability of pharmaceutical drugs and the impact ofenvironmental factors is important in quality control.

Tips
Low wavenumber measurement
A rejection filter is used for removing the Rayleighscattering light during Raman measurement. If its
performance is bad, it may also exclude meaningful
peak in low wavenumber region.

In this section Raman measurement was performed on acommercially available tablet containing carbamazepine thatwas heated to 200 °C (Figs. 7 and 8). It was confirmed that form
II was generated by heating. Raman imaging measurement
could obtain the local crystal structure transition information thatcannot be confirmed in the observed view.

Since rejection filter with angle adjustment function
makes it possible to adjust the angle of filter properly
and finely, it can remove the Rayleigh scattering
light precisely, and enables to perform Raman
measurement in low wavenumber region (Fig. 9).

Fig. 9 Rejection filter with angle adjustment function.

50 µm

In addition, JASCO can provide a solution for very low
wavenumber measurement. It is possible to mount
up to 3 rejection filters, whose incident angle can be
adjusted finely (Fig. 10).

Fig. 7 Observation view (after heating).

50 µm

Fig. 10 Very low wavenumber measurement unit.

Fig. 8 Raman imaging measurement result (after heating).
(red: form III, green: form II, blue: excipients etc.)

Adjusting the incident angle of their filters finely
improve its performance to the maximum, andenables to perform Raman measurement in lower
wavenumber region (Fig. 11).
14.7 cm-1-14.7 cm-1

9.6 cm-1

Int.

60

40

-9.6 cm-1

20

0

-20

-40

-60

Raman Shift [cm-1]

Fig. 11 Raman spectrum of L-cystine.

39

RAMAN SPECTROSCOPY APPLICATIONS - ART SCIENCE -Non-destructive analysis of artworks andother cultural assets
In the study of archeology, art history and in the restoration of works
of art, it is important to analyze and determine the materials thatwere historically used as pigments. Raman spectroscopy is a useful
tool for non-destructive and qualitative analysis of archaeologicalartifacts and works of art; this is because the measurement range
is wider than IR spectroscopy, which enables the measurement of
not only organic pigments but also inorganic pigments that typicallyhave absorption peaks in the low wavenumber range below 400
cm-1.1)

When measuring the red pigment, the drift in baseline causedby fluorescence was observed even with excitation at 785 nm.
Selecting 1064 nm excitation (with InGaAs detector) markedly
reduced the background fluorescence (Fig. 1).

Excitation: 532 nm

This section introduces the analysis of pigments by Raman
spectroscopy.

Excitation: 633 nm
Int.

1) Borgia, I. et al., Journal of Cultural Heritage, 8, 65-68 (2007).

Excitation: 785 nm

Qualitative analysis of colorants
It is generally considered difficult to obtain good Raman
spectra of colored materials because of their strong fluorescentcharacteristics, but it can be avoided by selecting an excitation
wavelength that is not close to the absorption band in themolecule; this is because these pigments typically havestrong and sharp absorption peaks in the visible region. In thisexperiment, water paint pigments were used as samples, thesewere excited using lasers with 3 different wavelengths for acomparative study.
In the case of the red pigment, a spectrum with good S/N was
obtained when selecting a longer excitation wavelength (Fig.
1). For the blue pigment, good S/N was obtained with a shorter
excitation wavelength (Fig. 2). And for the green pigment, agood spectrum was obtained with 633 nm excitation (Fig. 3).

40

Excitation: 1064 nm
3100

2000

1000

150

Raman Shift [cm-1]

Fig. 1 Raman spectra of red pigment.

Generally, observed color is the complementary color to
the absorbed light (Fig. 4). In this example, the red pigment
radiates strong fluorescence when excited at 532 nm because
it absorbs selectively the light in the region between green andblue. However, the green pigment that was expected to absorb
red light actually shows the best S/N when excited using 633
nm in the red region. The cause of this phenomenon may be
because the Raman peak of phthalocyanine, a component in
the green pigment, is selectively enhanced by the red laser due
to resonance Raman.

Combined analysis of toner pigment
Combining analysis using Raman spectroscopy with otherspectroscopic techniques yields complementary information.
This section demonstrates examples of combined analysis
using IQ Frame (refer to page 33).

Excitation: 532 nm

As an example, a cyan toner that was set in a diamondcompression cell was measured (Fig. 5).

Int.

Excitation: 633 nm
Excitation: 785 nm
3700

3000

2000

1000

150

Raman Shift [cm-1]

Fig. 2 Raman spectra of green pigment.

Fig. 5 Observation view (left) and diamond cell (right).
Excitation: 532 nm

Int.

First, the sample was measured using a UV-visible microspectrophotometer (Fig. 6), and a color calculation was
performed to accurately determine the the color of the toner(Fig. 7).

Excitation: 633 nm
50

Excitation: 785 nm
3700

3000

40
2000

1000

150

Raman Shift [cm-1]

Fig. 3 Raman spectra of blue pigment.

30
%R

20

10
0
200

400

600

800

Wavenumber [nm]Fig. 6 Absorption spectrum of cyan toner.
Fig. 4 Wheel of hue.
60

In addition, the spectral shapes of the green and blue samples
differ according to excitation wavelength; this is because thegroup vibration of the chromophore is caused by absorption
selectively enhanced by resonance Raman.
Library search of the database does not yield any spectra matchfor these pigments. This is because the ratio of peak intensity
is different depending on the excitation wavelength making
the spectra different from the library data previously obtained
from FT-Raman (1064 nm excitation). In addition, pigmentcomponents often contain various crystal polymorphs, sincephysical characteristics such as hue and corrosion resistancedepend on the presence of different crystal polymorphs,
Raman spectroscopy is proven to be a useful analytical tool toidentify these.

100

50
L*

80

60
b*

0
40

20
-50
-60
-60

0

50 60

0

a*

Fig. 7 Color calcuation result.

(Raman application data: 040-AN-0004)

41

Next, the sample was measured using IR microscopy, anda library search was performed using KnowItAll (Fig. 8). IR
microscopy was able to identify the main component (resin)
including the inside of the sample.
Epoxy-modified alkyd resin

Chemical analysis of archeological samples is very importantin the investigation of historical significance, but often artifacts
cannot be moved from their location for analysis. When in-situ
measurement is required for non-destructive analysis, a Ramansystem with a fiber probe is extremely convenient. This section
shows the identification of pigments used on a wall fresco.
The sample, a wall fresco was painted in the 16th century in asmall church located in northern Italy (Fig. 10). From the Raman
spectra, the components in the colored pigments (black, red,
blue and white) were identified as carbon, red ochre, azurite
and calcite, respectively (Fig. 11).

IR spectrum

3000

Non-destructive analysis of archaeological art

2000

1000

0

Raman Shift [cm-1]

Fig. 8 IR spectrum and library search result.

Finally, the sample was measured by Raman microscopy, anda second library search was performed using KnowItAll (Fig. 9).
Since the Raman measurement was performed directly in the
diamond cell, it must be noted that there is a diamond peak
present in the Raman spectrum. The pigment on the sample
surface was identified from the Raman spectrum.

Peak position of
diamondPhthalocyanine pigment

Fig. 10 Wall fresco.

Raman spectrum

3000

2000

1000

0

Raman Shift [cm-1]

Fig. 9 Raman spectrum and library search result.

Fig. 11 Measurement result.

We would like to express our sincere gratitude to Prof.
Sgamellotti of University of Perugia, Italy for providing the data.

42

RAMAN SPECTROSCOPY APPLICATIONS - FORENSIC SCIENCE -Forensic analysis of vermillion ink
Raman spectroscopy is used to analyze molecular structure frommolecular vibrations, similar to IR spectroscopy. There are several
distinct advantages of Raman spectroscopy, a smaller microscopic
area, spectra in the low wavenumber region can be easilyacquired, the sample can be measured using a non-destructive
and non-contact method, and precise information about theinside of a sample can be selectively obtained. For these reasons,Raman spectroscopy has become a key analytical technique inmany applications, such as semiconductors, pharmaceuticals,
and general chemistry. Recently, Raman is being more widelyused in the field of criminal investigations. For example, Raman
spectroscopy can be used in the examination of minute fragmentsof evidence and moreover, in the identification of poisonous or
toxic substances and illicit drugs without opening the container.
The following points should be noted for forensic investigation.
The first is interference from sample fluorescence. The mosteffective method to minimize fluorescence is selecting a laser
wavelength that does not cause fluorescence; generally, longer
wavelengths such as 785 nm or 1064 nm will result in a significant
reduction in fluorescence. A second technique to reduce
fluorescence is narrowing down the laser spot size or using anapertured confocal optical system, the spatial resolution in the xydirection (and even the z-direction) can be improved, minimizing
the influence of fluorescence from the matrix surrounding the pointof measurement. The second issue is the potential for damage tothe sample caused by laser irradiation. To avoid damage to thesample, it is important to set the optimum laser intensity by using
an attenuator.

In this section we used Raman spectroscopy to analyze seal
imprints. A Vermillion ink pad containing inorganic pigments suchas cinnabar and other types of ink pad containing organic pigments
are often used for seal imprints. These materials can be easily
discriminated using Raman spectroscopy in the low wavenumber
range. If the ink material can be characterized, it would be usefulin improving investigative results.

Measurement using a 532 nm laser
Seals imprinted on three different papers were measured using
a 532 nm laser (Figs. 1 and 2).

Fig. 1 Observation image of the seal.

43

In order to avoid damage to the sample, the laser intensity
was gradually increased until optimal. The fluorescence fromvermillion ink or its surrounding matrix can be very strong whenmeasured without using a confocal optical system, this makes
it difficult to detect any Raman peaks. In this measurement, aconfocal optical system was used and Raman measurement
could be made because of the improved spatial resolution.
From the results, Raman peaks were observed in the spectrumof Seal A, though it was affected by fluorescence. In the
spectrum of Seal B, some small peaks were just observable,
but it was impossible to detect Raman peaks in the spectrumof Seal C.
(Raman application data: 040-AN-0009)

Seal A
Seal B
Seal C

Analysis of the results
Taking a closer look at the results, the three spectra (Seal A: 532
nm excitation, Seal B and C: 785 nm excitation) were processedusing fluorescence correction and normalization in the analysis
software (Fig. 4). The Raman spectrum (2000–600 cm-1) ofeach sample was different and could be discriminated. Eachink sample has resin with the organic pigment. However, fromthe differences in the low wavenumber range (red frame) where
the peaks due to inorganic substances are expected to appear,
several peaks were barely seen in the spectrum of a Seal A as
compared with the spectra of Seal B and Seal C. Therefore, it
can be assumed that organic pigments are used primarily in ink
of Seal A, while Seal B and Seal C contain inorganic pigmentin the resin of the vermillion ink pad. If a library of reference
spectral for different types of vermillion ink pad and otherinks was available, imprints can be analyzed and identified bycomparing the obtained spectra of imprint with this reference
data.

Int.

Seal A

3200

2000

1000

200

Int.

Seal B-1

Raman Shift [cm ]Fig. 2 Raman spectra with excitation wavelength at 532 nm.
Seal C

Measurement using a 785 nm laser

2000

When the optimum conditions are selected, such as a suitablelaser wavelength, better spatial resolution and correct laser
intensity, good Raman spectra can be obtained even for samples
like vermillion ink that has a lot of background fluorescence.
(Raman application data: 040-AN-0009)

Seal A
Seal B
Seal C

Int.

2000

1000

Raman Shift [cm-1]

Fig. 3 Raman spectra with excitation wavelength at 785 nm.

44

1000

500

200

Raman Shift [cm-1]

To reduce the interference from fluorescence, Raman
measurements were made using a 785 nm laser (Fig. 3).
Raman peaks were observed in the spectrum of Seal A, but
fluorescence was still dominant. The fluorescence was greatly
reduced in the spectra of Seal B and Seal C, compared to the
532 nm laser.

3200

1500

200

Fig. 4 Spectra after fluorescence correction (normalized view).

As mentioned above, in Raman spectroscopy measurement ismade using a non-contact method with very simple operation;
this makes it a powerful tool when the sample is limited andnon-destructive testing is required.
(Raman application data: 040-AN-0009)

RAMAN SPECTROSCOPY APPLICATIONS - FORENSIC SCIENCE -Commercial pen ink identification in a
handwriting document
In Raman spectroscopy, spatial resolution is a key factor in
obtaining well discriminated data without mixing information fromother components. Since the performance of a confocal opticalsystem affects the spatial resolution, achieving good data depends
on the instrument design and quality of the optical system.
To demonstrate what can be achieved using Raman measurement,
this section shows the analysis of six types of commercial pen ink
in a handwritten document.

1

2

3

4

5

6

Raman spectra of each pen
Six different types of commercial pen ink used in handwritten
documents were measured to verify the possibility ofidentification (Figs. 1, 2 and 3).

Fig. 2 Observation view.

1
1

2

3

4

5

2

3

4

5

6

6

Fig. 1 Six types of commercial pen (left)
and their ink on handwritten document (right).

Sample #2 was measured with a 785 nm excitation because theinfluence of fluorescence was too strong with 532nm excitation.
In the measurement of all samples, Raman spectra could
be obtained without the influence of the paper (cellulose).
Raman spectra of sample #3 could not be obtained because
the influence of fluorescence was too strong even at 785 nm
excitation.

45

Int.

Sample #1

Excitation: 532 nm

Sample #2

Excitation: 785 nm

Sample #4

Excitation: 532 nm

Sample #5

Excitation: 532 nm

Ink components of the vertical lines (second stroke) were detected
at each intersection, and it means that Raman spectroscopy could
selectively detect the surface information.
The results indicate that Raman spectroscopy is a useful tool todetermine the order of a handwriting intersection.

Int.

Sample #6

2000

1500

1000

Excitation: 532 nm

500

150

Raman Shift [cm-1]

Fig. 3 Obtained Raman spectra.
(After fluorescence correction, Y-axis offset)

After registering reference data of each product in KnowItAll, alibrary search can be made to identify unknown pen ink samples.

2000

1500

1000

500

150

Raman Shift [cm-1]

Fig. 5 Raman spectra of intersetions.
green: black pen #1 (vertical line) and black pen #4 (horizontal line)
blue: black pen #1 (vertical line) and black pen #6 (horizontal line)

Determining the stroke order of intersections using
Raman spectroscopy
Using three different black pens (#1, #4 and #6), vertical lines
were drawn after drawing horizontal lines (Fig. 4), and the six
intersections were measured to see if the stroke order could bedetermined (Figs. 5, 6 and 7).

Int.

2000

1500

1000

500

150

Raman Shift [cm-1]

Fig. 6 Raman spectra of intersetions.
green: black pen #4 (vertical line) and black pen #1 (horizontal line)
blue: black pen #4 (vertical line) and black pen #6 (horizontal line)

Int.

2000

1500

1000

500

150

Raman Shift [cm-1]

Fig. 4 Observation view of intersection.
(red arrow: first stroke, blue arrow: second stroke)46

Fig. 7 Raman spectra of intersetions.
green: black pen #6 (vertical line) and black pen #1 (horizontal line)
blue: black pen #6 (vertical line) and black pen #4 (horizontal line)

Tips
Spectral search program - KnowItAll WILEY's KnowItAll, which is a highly regarded data
search software, is used as standard. The search
software starts up with one click from the analysis
program (Fig. 8).

KnowItAll includes various functions such as spectral/peak/structure search, mixture analysis, spectralinterpretation support and more. In addition, 645
organic compound spectra and 650 JASCO original
spectra are installed as standard as a reference library.
WILEY's 25,000 Raman spectra are also available asan option.
Mixture analysis
For unknown samples containing multiple components,
up to five components can be identified from one
spectrum.
Spectral interpretation support (AnalyzeIt™ Raman)
This function can find the functional groups that match
the peaks of spectrum.

One-click transfer

User database builder
Creating a user-defined database, it can register
the sample spectra, structural formula and physicalproperties. It enables to unitarily manage and sharethe measurement data.
Multi-technique simultaneous spectral searching
KnowItAll's multi-technique simultaneous spectral
searching allows to search the IR and Raman spectra
simultaneously and find the most relevant hits in eachdatabase linked to each other by chemical structure.

Fig. 8 KnowItAll.

47

JASCO CORPORATION

2967-5, Ishikawa-machi, Hachioji-shi, Tokyo 192-8537 Japan
Tel: +81-42-649-5177 Fax:+81-42-646-4515 Web: www.jasco.co.jp
Japan

JASCO INTERNATIONAL CO., LTD.

11-10, Myojin-cho 1-chome, Hachioji-shi, Tokyo 192-0046, Japan
Tel: +81-42-649-3247 Fax: +81-42-649-3518 Web: www.jascoint.co.jp/english/
Australia, Hong Kong, India, Indonesia, Korea, Malaysia, New Zealand, Pakistan,Philippines, Russia and CIS countries, Singapore, Taiwan, Thailand, Vietnam

JASCO INCORPORATED

28600 Mary’ s Court, Easton, Maryland 21601, U.S.A.
Tel: +1-410-822-1220 Fax: +1-410-822-7526 Web: www.jascoinc.com
Argentina, Bolivia, Brazil, Canada, Chile, Colombia, Costa Rica, Guatemala, Mexico,
Paraguay, Peru, Puerto Rico, United States of America, Uruguay, Venezuela

JASCO EUROPE S.R.L.

Via Luigi Cadorna 1, 23894 Cremella (LC), Italy
Tel: +39-039-9215811 Fax: +39-039-9215835 Web: www.jasco-europe.com
JASCO Deutschland www.jasco.de | JASCO UK www.jasco.co.uk | JASCO France www.jascofrance.fr
JASCO Benelux www.jasco.nl | JASCO Spain www.jasco-spain.com
Algeria, Austria, Belgium, Cyprus, Denmark, Egypt, Finland, France, Germany, Greece, Hungary,
Israel, Italy, Jordan, Kuwait, Luxembourg, Morocco, Netherlands, Norway, Poland, Portugal, Romania,
Saudi Arabia, South Africa, Spain, Sweden Switzerland, Tunisia, Turkey,
United Arab Emirates, United Kingdom, Yemen
ISO-9 0 0 1

JASCO CHINA (SHANGHAI) CO., LTD.

Room No.D, 10F, World Plaza, 855 Pudong South Road, Pudong New Area, Shanghai, China
Tel: +86-21-6888-7871 Fax: +86-21-6888-7879 Web: www.jasco-global.com
China

ISO-14001

CERTIFIED

Products described herein are
designed and manufactured by
ISO-9001 and ISO-14001 certified
JASCO Corporation
e6712-2406ENG

The contents of this brochure are for reference and illustrative purposes only. Information, descriptions, and specifications in thispublication are subject to change without notice. JASCO assumes no responsibility and will not be liable for any errors or omissions
contained herein or for incidental, consequential damages or losses in connection with the furnishing, performance or use of this material.