Application eBook
Fluorescence Spectroscopy

Table of Contents
Introduction
Principle
				Instrument Performance			Applications - Life science - 			
Applications - Material science - 		Applications - Environmental science -

2

3
6
8
24
38

What is fluorescence?
Some material has the property that the light can be given by the absorbed energy. This phenomenon is
generally called "luminescence". Table 1 shows the types of luminescence. Fluorescence is the one ofphotoluminescence, which is the light emission after the absorption of photon (ultraviolet/visible light).
Table 1. Types of luminescence
Type

SourceExampleElectroluminescenceElectric field

LED, organic EL, inorganic EL

Chemical luminescence

Chemical reaction,oxidation

White phosphorus (blue-green light emission),formaldehyde, paraldehyde with alkali/ethanol
solution, oxidation of siloxene by Ce (IV)

Photoluminescence

UV rays, visible rays,
infrared rays

Fluorescence and phosphorescence

Bioluminescence

Physiologicalabsorption, enzymaticoxidation

Fireflies produce luminescence by catalytic oxidationof luciferase

Radioluminescence

α/β/γ rays, neutrons

Scintillator (NaI · TII)

X-ray luminescence

X-ray

X-ray phosphor(e.g. ZnCdS: Ag, ZnS: Ag)

CathodoluminescenceElectron beam

Phosphor, cathode ray tube for CRT, apatite

Thermoluminescence

Heat

Luminescence due to temperature rise (fluorite, sulfide
phosphor)

SonoluminescenceSound waves,ultrasonic waves

Chemical reaction by the stimulation of sound(oxidation reaction of luminol)

Triboluminescence

Friction, strain anddestruction

Crystal sugar, ZnS

Absorption and emission
The energy contained in a photon is expressed by E = hν (E: energy,h: Planck’s constant, ν: frequency). When light hits a substance, its
energy is absorbed by electrons, and the electronic configuration
changes from the stable ground state to a higher energy excitedstate. Luminescence occurs when this energy is released in theform of light emission rather than molecular vibrations or heatgeneration. The electronic configuration then returns to theground state (Figure 1). In many cases, part of the absorbed energy
is emitted as thermal energy, so that the emitted light has a longer
wavelength than the absorbed light (Stokes shift).

Vibrational relaxation(Heat etc.)
Excited state

Heat

EnergyFluorescence

E = hvGround state

Figure 1. Jablonski diagram showing
types of light emission
3

Excitation spectrum and fluorescence
spectrum
In the excitation spectrum, the emission wavelengthis fixed, and the excitation wavelength is scanned tomeasure the fluorescence intensity. The excitation
wavelength that gives the maximum fluorescenceintensity is the optimal excitation wavelength. The
shape of the excitation spectrum is similar to that of
the absorption spectrum (Figure 2), and the strongest
fluorescence is also given at maximum absorption
wavelength. It occurs because the amount of excitation
light, which is absorbed at maximum absorption
wavelength, is the largest, and the emitted energy whentransferring from excited state to ground state is large.
1000

Int.

600

400

200

0
320

350

400
Wavelength (nm)450

Excitation spectrum

A.U.

300

350

400

Wavelength (nm)Figure 2. Absorption spectrum and excitation
spectrum

The fluorescence spectrum is acquired by fixing theexcitation wavelength and scanning the emission
wavelength. Since the fluorescence spectral profiles
do not vary with excitation wavelength (Figure 3), theoptimal excitation wavelength that gives the strongest
fluorescence light should be used for the fluorescencemeasurement. The peaks of fluorescence spectrum
appear at longer wavelength than the ones ofabsorption spectrum. Since the part of emitted energy
is lost by heat etc, that phenomenon occurs.

323 nm
339 nm
356 nm800

Absorption spectrum

500

Figure 3. Fluorescence spectra at different excitation
wavelengths
Most of phosphor (benzene, anthracene, rhodamine 6G etc.) has the mirror image relationship between
absorption and fluorescence spectra (Figure 4 - right) because the interval of vibrational level in its excitedstate is similar to the one in its ground state (Figure 4 - left). Sometimes, its mirror image relationship may not
be satisfied due to the interaction between sample and solvent.

S1
3

2

Energy

0

S0

1

2

3

4

3
4

0

0

0

0

1

0

0

0

0

0

0

1

2

0

3

0

4

0

Nuclear configuration

Figure 4. Vibration levels in excited and ground states (left), mirror image relationship between
absorption and fluorescence spectra (right)
4

Fluorescenceintensity

2

Absorbance1

4Advantages of fluorescence spectroscopy

Fluorescence spectroscopy offers the following advantages:● High sensitivity
Absorption spectroscopy detects decreases in incident light intensity, whereas fluorescence spectroscopy
detects the intensity of emitted light. It is highly sensitive because it detects the difference from zero lightintensity.
● High selectivity
Molecular species that emits the fluorescence are limited. In addition, emission wavelength and excitation
wavelength depend on the phosphor. Therefore, fluorescence spectroscopy is the analytical method withhigh selectivity. This advantage is used for the fluorescence labeling to monitor the target molecule bybinding it to the fluorescent molecule.
● High environmental dependency
The shape and intensity of the fluorescence spectrum are sensitive to the environment surrounding thefluorescent molecules (solution pH, temperature, solvent type, coexisting salt). This can be used to probe
the surrounding environment.

Solutions by fluorescence spectroscopy
Fluorescence spectroscopy is applicable at various fields and purposes, and provides the breakthroughs as
below.
Life science• Sample concentration
It indicates the concentration information of fluorescent in sample. It can also show the concentrationof target molecules by specifically binding/interacting with fluorescent dyes (or fluorescent probes).
• Fluorescence anisotropy/Fluorescence polarization
It indicates the degree how much the fluorescence light which is emitted from fluorescent ispolarized. It can also detect the change of protein structure, the change of molecular weight at
decomposition of biopolymer, and the interaction between biomolecules (protein-protein, antigenantibody, DNA-protein, protein-protein).
• Melting temperature
It indicates the temperature when the abundance ratio of native protein is equal to the one ofdenatured protein, and also indicates thermal stability of protein.
• Dissociation constantIt represents the concentration ratio between the complex and its component molecules at chemical
equilibrium. It can be used as the parameter of the affinity between protein and ligand.
Material science• Fluorescencee property
It shows specific excitation/emission information (wavelength and intensity) which the fluorescent has.
• Quantum yield
It is the ratio between photons which is absorbed to sample and the ones which is emitted fromsample. It can express the emission efficiency of fluorescent.
• Phosphorescence lifetime
It is the time when the amount of fluorescent molecules in excited state is decreased to 1/e(approximately 37 %). It can provide the meaningful information about emission mechanism.
• Luminous color/Color rendering index
Luminous color can be represented by quantifying the fluorescent's color. Color rendering index
indicates the degree how much the color appearance of objects under the light source reproduces
the one under sunlight.

5

FP-8050 Series Spectrofluorometer
Outstanding sensitivity and linear dynamic range

The high-throughput optical system and low noise signal processing of the FP-8050 Series results in a highsignal-to-noise (S/N) performance up to 8,500:1* (RMS). In addition, the dynamic range of FP-8050 Series has
been developed to provide up to 7 orders of magnitude. As shown in the calibration curve for fluorescein,there is excellent linearity even for samples at very low concentrations.
90

−
−
−
−
−

Correlation coefficient = 0.9998
2

10

Int.

Int.

60

104

1×10−11 mol/L
5×10−12 mol/L
1×10−12 mol/L
5×10−13 mol/L
1×10−13 mol/L

40

100

10-2

20

0
500

510

520
530
Wavelength[nm]

540

10-4

550

10-12

10-10

10-9

10-8

10-7

10-6

Concentration [mol/L]

10-13

Fluorescein spectra for a range ofconcentrations

10-11

Calibration curve for fluorescein(Auto Sensitivity Control System (SCS) ON)

Higher-order light cut-off filters

In order to remove peaks originating from higherorder diffracted light, cut-off filters appropriate to
the measurement wavelength should be used. The
models FP-8350, FP-8550, and FP-8650 include
cut-off filters that are switched automatically as the
wavelength range is set by the user.

Artificial peaks
from second orderdiffracted light

Without Cut-Off Filter
With Cut-Off Filter
3D Spectra of Fluorescent Orange Color Plate

Auto-adjustment of detector gain and sensitivity

The FP-8050 Series includes both Auto-Gain and Auto Sensitivity Control System (SCS), which automaticallyadjust the detector gain and sensitivity for optimum measurement. Auto-Gain automatically adjusts the gainof the signal from the detector so that the S/N is optimized throughout the entire scan range for spectral
measurement, and weak fluorescence peak shapes can be observed against scattered light with a high S/N.
The Auto-SCS allows the user to create calibration curves for a wide concentration range without having to
manually change the instrument sensitivity settings.
1.5

600

− Auto-Gain ON− Auto-Gain OFF

1

Int.

Int.

400

200

0

0.5

Scattering light
Fluorescence

300

400

500
600
Wavelength[nm]

0

700

750

-0.5
500

600
Wavelength[nm]Fluorescence spectra of quinine sulfate solution*Typical S/N for FP-8500 for water Raman, using on peak baseline noise.
6

700

750

Accurate spectral correction

Spectral correction should be made to any fluorometer for themeasurement of accurate and reliable spectral data. All models
in the FP-8050 Series can be spectrally corrected using asimplified procedure. JASCO has a range of calibration standards
for spectral correction, including standard light sources andRhodamine B.
Calibrated WI light source

User-friendly interface

A simple sequence of buttons guides the user through routine
operation from measurement to saving data.

Enhanced measurement functions- Saturation log (photometric value)- Fluorescence Maxima search- Automated shutter function (open/close)- Self-motion auto-zero- Simple parameters

Automatic Accessory Recognition

Accessories are automatically recognized when installed in the instrument. Spectra Manager™ logs theaccessory name and serial number; this information is saved in the data file for a complete record of themeasurement.

Daily monitoring of instrument performance
Validation program
The Validation program includes a full suite of tests to validate instrument performance. When
executed, simple prompts guide the user on how to perform the tests. All instruments in the
FP-8050 Series include a mercury lamp as standard for wavelength calibration.
Daily check program
The Daily Check is a simple alternative to the Validation program; a less comprehensive
performance check can be made at any time for continuous performance monitoring. After
starting the Daily Check program, a timer is activated, and sample measurement is executed
automatically after a predetermined stabilization time. The batch display lets the user check the
cumulative data over a period of time.

Instrument activity log

The instrument activity log is a useful alternative to the paper log books found in multi-user facilities. A record
is maintained of user, duration of operation and lamp usage.
7

LIFE SCIENCE APPLICATIONS

Fluorescence measurement ofheat-denatured protein
In order to evaluate the stability of protein, measuring the fluorescence that derives from aromatic amino acid(side-chain) is effective. Aromatic amino acids of protein are phenylalanine, tryptophan and tyrosine. They havethe maximum fluorescence at 282 nm, 304 nm and 348 nm respectively (aqueous solution), and native protein
has the maximum fluorescence at 340 nm because the fluorescence peaks of aromatic amino acids are stacked.
Ramping the temperature of native protein, the high order structure varies by thermal denaturation. It meansthat the location of aromatic amino acid is changed from inside of protein to outside, and the maximum emission
wavelength is shifted to long wavelength due to the change of the interaction between side-chains. Therefore,monitoring the maximum emission wavelength by changing the temperature or pH of protein enables to evaluate
the structure stability of protein.

: Aromatic
amino acid
Native protein

Denatured protein

Since the structure stability of protein is directly linked to its functional stability, the thermal stability evaluationof protein by fluorescence measurement will be excellent solution at the following situations in R&D of antibody
and diagnostic drugs.
- Stability comparison between before and after modified protein- Stability comparison between original biological drug and biosimilar drug
- Investigation of drug storage condition
JASCO can provide the peltier cell holder, which can control the temperature of liquid sample with high accuracy.
In addition, it is capable of the automatic measurement at a specific temperature and melting temperature (Tm)analysis.

8

App. Note: 210-FP-0002

Fluorescence Measurement of Heat-Denatured Lysozyme
Thermal denaturation of lysozyme which includes the tryptophan residue was evaluated.
[Sample]- Lysozyme aqueous solution: 0.1 mg/mL[Measurement parameters]
Bandwidth (excitation):		
2.5 nmBandwidth (emission): 		
5 nm
Response: 				0.5 sec.
Sensitivity:				Medium
Measurement start temperature:
15 °C
Measurement end temperature:
90 °C•

Excitation wavelength:		
Wavelength range (emission):
Data interval: 			
Scanning speed:			Temperature interval:		Temperature gradient:		

280 nm290–450 nm
0.2 nm
200 nm/min.
5 °C
1 °C/min.

In order to make the sample temperature uniform, the measurement was performed during stirring thesample with stirrer.
Spectra measurement was performed after 60 seconds from reaching to the setting temperature.

•

Figure 2a shows the changes of fluorescence spectra related to the temperature variations, and figure 2b shows
the ratio between the fluorescence intensity at 340 nm and the one at 350 nm. As shown in figure 2a, the peakwavelength was shifted from 340 nm to 350 nm while the temperature was raised. Plotting the fluorescenceintensity ratio (350 nm/340 nm) realized that its ratio was changed significantly at approximately 70 °C. These
results indicates that the protein was denatured by raising temperature, and that tryptophan residue which wasburied in protein was exposed to outside of protein. In addition, it estimated the melting temperature of lysozyme
at approximately 70 °C.
a. Changes of fluorescence spectrab. Temperature monitoring of fluorescenceintensity ratio (350 nm/340 nm)

700

Int.

400

200

0

90 °C
300

350
Wavelength (nm)

400

450

Fluorescence intensity ratio (350 nm/340 nm)

15 °C
600

1.08
1.06
1.04
1.02
1
0.98
0.96
0.94
0.92

10

30

50
Temperature (ºC)

7090

Figure 2. Measurement results
9LIFE SCIENCE APPLICATIONSMonitoring of protein behavior
by using FRET
FRET is the mechanism that the energy is transferred non-radiatively from an excited state donor to aneighbor acceptor (Figure 1). FRET is an abbreviation for Forester (or fluorescence) resonance energy
transfer. Since FRET typically occurs when the distance between the donor and acceptor is 1–10 nm,
it enables to monitor the structure change of protein and the interaction between specific proteins bybinding donor and acceptor to the target protein.

Relaxation
Relaxation
Non-radiative
energy trasnfer
Non-radiative
energy trasnfer

Fluorescence
FRET
Absorption

Fluorescence

Donor

Acceptor

Figure 1. Energy transition in a FRET pair
FRET is monitored by the spectrofluorometer, whichmeasures the fluorescence/quenching of acceptor
or excited donor. FRET efficiency depends on thefollowing factors (Figure 2).
1. Spectral overlap between the donor and acceptor
As the overlap area of the donor's fluorescence
spectrum and the acceptor's absorption spectrum
is larger, FRET efficiency is higher.
2. Distance between donor and acceptor
FRET efficiency is inversely proportional to the six
power of distance between the donor and acceptor.
3. Orientation of donor and acceptor
FRET efficiency is a maximum when the two dipole
moments are parallel or anti-parallel to each other,and no energy transfer occurs when the dipole
moments are perpendicular to each other.

1. Spectral overlap between donor and acceptor
Donor excitation
spectrum

Donor fluorescence
spectrum
Acceptor absorption
spectrum

wavelength (nm)2. Distance between donor and acceptor

Donor

FRET

Acceptor

3. Orientation of donor and acceptor

Donor

Acceptor

FRET: Dipole moment

Figure 2. Factors that influence FRET
10

One of monitoring ways of protein behavior by using FRET is a cyan
fluorescent protein (CFP) - yellow fluorescent protein (YFP) pair.
CFP and YFP, which are color variants of green fluorescent protein(GFP), are well-known as the fluorescent protein that FRET occurs. CFP
absorbs excitation light of 433 nm, and produces fluorescence light of
476 nm. When CFP is close to YFP, the energy which CFP has absorbed
is transferred to YFP by the interaction between CFP and YFP, and YFP
produces fluorescence light of 527 nm (Figure 3) [1].

433 nmCFP
476 nm433 nmIts property can be applied to protein behavior monitoring by binding
CFP and YFP to the target protein.
Fusing CFP and YFP to two different host proteins enables to monitor theprotein-protein interactions (Figure 4. intermolecular FRET), and fusing
CFP and YFP to two different sites in the same protein enables to monitor
the protein conformational change (Figure 4. intramolecular FRET).
[1] Roger Y. Tsien 2009 Constructing and Exploiting the FluorescentProtein Paintbox (Nobel Lecture). Angew. Chem. Int. Ed. 48: 5612–5626

YFP

FRET
YFP

CFP
476 nm

527 nmFigure 3. FRET between CFP and
YFP

FRET

CFP

CFP

FRET

CFP

YFP

YFP

CFP

YFP

YFP

Figure 4. Intermolecular FRET (left) and Intramolecular FRET (right)
λex

DNA aptamer

This property can be also applied to biosensor. One
of its applications is the biosensor by using DNA
aptamer, which has the capability to bind a specificmolecule.

Acceptor

λem

Donor

TargetMolecule

TargetMolecule

λex

λem

Figure 5. Schematic diagram of biosensor by using
DNA aptamer

DNA aptamer, whose ends are fused donor/acceptor
fluorescent molecules, can detect the target molecule
because FRET occurs by changing its structure whenbinding DNA aptamer to target molecule (Figure 5).

JASCO's spectrofluorometer can detect FRET phenomenon with high sensitivity, and provide its useful
information to the user.
11

LIFE SCIENCE APPLICATIONS

Sensitivity evaluation of biosensor
by using FRETOne of FRET application examples is the murine anti-TNT monoclonal antibody (anti-TNT mAb), which canform a complex with trinitrotoluene (TNT). Anti-TNT mAb absorbs the excitation light of 280 nm, and produces
the fluorescence light of 340 nm. When anti-TNT mAb forms a complex with TNT, FRET is occurred between
the anti-TNT mAb (donor) and TNT (acceptor). The energy is transferred from the anti-TNT mAb excited at
280 nm to the TNT, and the fluorescence at 340 nm from the anti-TNT mAb is quenched (Figure 1). By utilizing
this interaction as biosensor, this article shows the evaluation result of anti-TNT mAb sensitivity against thearomatic nitro compound as well as TNT by using spectrofluorometer.
λex = 280 nm

λem = 340 nm

λex = 280 nm

TNT

λem = 340 nm
Quenching by
FRET

Figure 1. Schematic illustration of fluorescence quenching by FRET
[Reagents and samples]- Murine anti-TNT monoclonal antibody (anti-TNT mAb): 1.6×10-7 mol/L[1]- Aromatic nitro compounds
Trinitrotoluene (TNT):		4.4×10-8, 4.4×10-7, 4.4×10-6.5, 4.4×10-6, 4.4×10-5.5, 4.4×10-5 mol/L
Trinitrobenzene (TNB):		4.7×10-8, 4.7×10-7, 4.7×10-6.5, 4.7×10-6 mol/L
2,6-dinitrotoluene (2,6-DNT): 5.5×10-8, 5.5×10-7, 5.5×10-6, 5.5×10-5, 5.5×10-4 mol/L
2-Nitrotoluene (NT):		7.3×10-8, 7.3×10-7, 7.3×10-6, 7.3×10-5, 7.3×10-4 mol/L
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX): 4.5×10-8, 4.5×10-7, 4.5×10-6, 4.5×10-5, 4.5×10-4 mol/L- SolventpH 7.4 phosphate buffer[1] Satoko Suzuki, Toshifumi Uchiyama, Ken-ichi Akao, Koushi Nagamori, Hiroshi Nakayama andYuji Ito, TNT sensing using non-labeling FRET, Japan-Taiwan Medical Spectroscopy International
Symposium/14th Annual Meeting of the Japan Association of Medical Spectroscopy, AWAJI, JAPAN2016[Measurement flow]
4.4×10-8 mol/L TNT solution was added to 800 µL of 1.6×10-7 mol/L anti-TNT mAb, and the fluorescencemeasurement was performed. After measurement, the final concentration of TNT was increased step-by-stepby adding each TNT solution (4.4×10-7 mol/L, 4.4×10-6 mol/L, 4.4×10-5.5 mol/L and 4.4×10-5 mol/L), and fluorescencemeasurement of each different TNT concentration solution was performed.
Regarding the other aromatic nitro compounds, similar measurements were performed.

12

App. Note: 210-FP-0022[Results]
The fluorescence spectra of mixture solution of anti-TNT mAb and TNT were shown in Figure 2. As the TNT
concentration increases, the fluorescence intensity of anti-TNT mAb becomes weak. Similar measurements were
performed using other aromatic nitro compounds as samples. The degree of progress of complex formation reaction
was determined by the decrease of the fluorescence intensity at 340 nm. Figure 3 shows the relations between thedose of aromatic nitro compounds and the reaction ratio. From this curve, the half maximal inhibitory concentration(IC50) was estimated (Table 1). This result shows that anti-TNT mAb has sensitivity to TNT, TNB, NT and 2,6-DNT,
especially have high sensitivity to TNT and TNB whereas cannot detect RDX.
From these results, anti-TNT mAb is expected as high sensitive nitro compounds biosensor without using fluorescentlabel.
1500
TNT (Final concentration)
0 mol/L
4.4 × 10 -10 mol/L
4.8 × 10 -9 mol/L
1.9 × 10 -8 mol/L
6.3 × 10 -7 mol/L
5.0 × 10 -6 mol/L

1000
Int.

Table 1. Analysis of binding of different explosive
compounds to anti-TNT
Name

Structure

IC 50 (M)

CH₃

TNT

NO2

O2N

4.0E-08

NO2

500

NO2

TNB

3.8E-08
NO2

O2N
CH₃

0
300

350

400
Wavelength (nm)

450

500

2,6-DNT

O2N

NO2

7.9E-06

NO2

3.6E-04

CH₃

Figure 2. Dose-response curve of Anti-TNT mAb andaromatic nitro compounds

NT
O2N

RDX

N

N
N

NO2

-

NO2

120

100

Non-reaction rate (%)

80

60

40
TNT
TNB
2,6-DNT

20

NT
RDX

0
1.E-11

1.E-09

1.E-07
Final concentration (mol/L)

1.E-05Figure 5. Dose-response curve of Anti-TNT mAb andaromatic nitro compounds
13LIFE SCIENCE APPLICATIONS

Fluorescence depolarization
measurement
Fluorescence anisotropy (r) is the ratio of the polarized component to total intensity. It depends on the
fluorescence lifetime (the time from exciting the phosphor to emitting the light), and depends on what degree
its fluorescent does not move during the fluorescence lifetime and maintains the polarization when it is excited.
Therefore, measuring its parameter (or fluorescence polarization) enables to know the degree of fluorescent’s
rotation, which depends on the molecular size, the viscosity around the molecule, the intermolecular interaction
and so on. These techniques mainly apply to the following measurements of bio-sample.
•••

Size/conformation monitoring of biopolymer
Evaluation of local structure in biopolymer
Evaluation of inter-biomolecule interaction

This application note will demonstrate the use of the spectrofluorometer and its automatic polarizers to monitor
the temperature-dependent polarization of diphenyl hexatriene (DPH) upon addition to a lipid bilayer.
DPH which is oriented between the phospholipid molecules has strong polarization property, and the status ofbiomembrane can be monitored by depolarization measurement.

DPH which is oriented
between the phospholipid
moleculeshydrophillic group
Hydrophobic group

DPH[Measurement procedure](1) Calculation of G factor
Since the monochromator in emission side has polarization characteristics,
G factor (G) for correcting the instrumental polarization artifacts should
be calculated beforehand by using fluorescent dye for fluorescence
depolarization measurement. The polarizer at excitation side should
be installed to horizontal orientation (H), and then the intensity when
the analyzer at emission side is set to vertical orientation (V) shouldbe measured (IHV), and the one when the analyzer is set to horizontal
orientation (H) should be measured (IHH). G factor is calculated as below.

G factor (G):
14

𝐺𝐺 =

𝐼𝐼𝐻𝐻𝐻𝐻
𝐼𝐼𝐻𝐻𝐻𝐻

Ex
IH

IHH
Em

IHV

Figure 1. Measurement overview of
G factor

App. Note: 210-FP-0016(2) Calculation of fluorescence anisotropy and fluorescence polarization
The polarizer at excitation side should be installed to vertical orientation(V), and then the intensity when the analyzer at emission side is set tovertical orientation (V) should be measured (IVV), and the one when theanalyzer is set to horizontal orientation (H) should be measured (IVH). G
factor is applied to IVH to perform the instrumental correction.

Ex

IV

G·IVH

Total fluorescence intensity (F):𝐹𝐹 = 𝐼𝐼𝑉𝑉𝑉𝑉 + 2𝐺𝐺 ∙ 𝐼𝐼𝑉𝑉𝑉𝑉
𝑟𝑟 =Fluorescence anisotropy (r):𝑃𝑃 =Fluorescence polarization (P):𝐼𝐼𝑉𝑉𝑉𝑉 − 𝐺𝐺 ∙ 𝐼𝐼𝑉𝑉𝑉𝑉
𝐼𝐼𝑉𝑉𝑉𝑉 + 2𝐺𝐺 ∙ 𝐼𝐼𝑉𝑉𝑉𝑉
𝐼𝐼𝑉𝑉𝑉𝑉 − 𝐺𝐺 ∙ 𝐼𝐼𝑉𝑉𝑉𝑉
𝐼𝐼𝑉𝑉𝑉𝑉 + 𝐺𝐺 ∙ 𝐼𝐼𝑉𝑉𝑉𝑉

Em

IVV

Figure 2. Measurement overview
of fluorescence anisotropy andpolarization[Results]
G factor was measured by using mixture sample (liposome and DPH), and then temperature scan measurement was
performed. DPH is located to the space between the lipid bilayers, and is oriented in a certain direction. Figure below
shows the fluorescence anisotropy at each temperature, and indicates that the motion of DPH is confined at low
temperature. In addition, it also shows that the depolarization occurs at approximately 40 ºC. It means that the fluctuationof layer became large due to phase transition of lipid bilayer, and the motion of DPH became large.

[Samples]- Phospholipid: 130 µM- DPH: 0.6 µM

0.4

0.3

r

0.2

0.1

0

25

30

40
Temperature (ºC)

50

54.7

Figure 3. Fluorescence anisotropy at each temperature(DPH interacting with liposome)[Measurement parameters]
Excitation wavelength:		
357 nmEmission wavelength:		
430 nmBandwidth (excitation):
5 nm
Bandwidth (emission): 		
5 nm
Response:			2 sec.
Data interval: 		
0.1 ºC
Ramping rate:			20 ºC/hour

15

LIFE SCIENCE APPLICATIONS

Enzyme kinetics probed byfluorescence spectroscopy
Enzyme activity is frequently investigated in the medicinal, biochemistry, and food science research fields toelucidate reaction rates and the affinity of enzyme-substrate interactions. There are some way to monitor theprocess of enzyme-substrate interactions, and spectrofluorometer is one of their effective tools.
JASCO offers the hardware/software for enzyme kinetics, which provides the reliable data. This section
introduces how to obtain enzyme kinetic data.
Overview
One of the best-known models of enzyme kinetics is Michaelis-Menten kinetics. It is the reaction model thatan enzyme (E) binds to a substrate (S) to form a complex (ES), which in turn releases a product (P) regenerating
the original enzyme.

EnzymeSubstrate

Enzyme (E) + Substrate(S)Enzyme-Substrate Complex (ES)The initial velocity (v) of formation of P is represented
as the following formula.
𝑣𝑣 =𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 × [𝑆𝑆]𝐾𝐾𝑚𝑚 + [𝑆𝑆]

Product

0.07

Vmax

0.06

(1)

This equation is called the Michaelis-Menten
equation. Here, [S] is the substrate concentration.
Vmax represents the maximum reaction velocity atsaturating substrate concentration, and Km , which iscalled the Michaelis constant, is equal to the substrateconcentration at which the reaction velocity is half ofVmax. Plot v versus [S] based on the equation (1) iscalled Michaelis-Menten plot (figure 1), which enables
to calculate the Vmax and Km.

Enzyme (E) + Product (P)v

Vmax /2

0.04

0.02

0

0

Km

1

2
[S]

3

4

Figure 1. Michaelis-Menten plot

The high Vmax means that the maximum activity of target enzyme is high. In addition, Km indicates the affinity
between enzyme and substrate. Low Km means that the affinity between enzyme and substrate is high, andenzyme-substrate complex is formed quickly. On the other hand, high Km means that the affinity between
enzyme and substrate is low, and enzyme-substrate complex is formed slowly. Vmax and Km, can be calculatedby the following procedure, which requires the analytical instrument such as the spectrofluorometer.
(1) Adds the substrate into enzyme solution, and perform time-course measurement, which monitors thefluorescence intensity of product or substrate.
(2) Obtains the initial velocity (v) by the slope at the start of reaction.
(3) Changes the substrate concentration, and obtains the initial velocity (v) through (1) and (2). And then, create[S] versus v plot.
16

Since obtaining V max and K m from Michaelis-Menten plot by eye makes the error easily, the modificationof equation (1) is performed. The following methods are well-known as the modification method ofMichaelis-Menten equation, and Vmax and Km can be obtained from slope and intercept of these plot.
Lineweaver-Burk plot: 1/[S] vs 1/v
1
𝐾𝐾𝑚𝑚
1
1
=
×
+
𝑣𝑣 𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 [𝑆𝑆] 𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚

230

(2)

Plot 1/v versus 1/[S] (Figure 2) based on the modified versionof the Michaelis-Menten equation given above. Determine theslope (a) and the intercept (b) of the straight line using the leastsquares method and then calculate Vmax and Km using equations(3) and (4), respectively. This plot is often used for calculating Vmax
and Km, and is effective at low substrate concentration range.
1
𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 =
(3)
𝑏𝑏

200

Slope = Km/Vmax
1/v

1/Vmax

100

0

-1

5

-1/Km

1/[S]

10

15

Figure 2. Lineweaver-Burk plot

(4)

𝐾𝐾𝑚𝑚 = 𝑎𝑎 × 𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚

0

Hofstee plot: [S] vs [S]/v
[𝑆𝑆]
1
𝐾𝐾𝑚𝑚
=
× [𝑆𝑆] +𝑣𝑣
𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚
𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚

Plot [S]/v versus [S] (Figure 3) based on the modified versionof the Michaelis-Menten equation given above. Determine theslope (a) and the intercept (b) of the straight line using the leastsquares method and then calculate Vmax and Km using equations(6) and (7), respectively. This plot is effective at high substrateconcentration range.
1
𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 =(6)𝑎𝑎
𝐾𝐾𝑚𝑚 = 𝑏𝑏 × 𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚

𝐾𝐾𝑚𝑚 = −𝑎𝑎

Slope = 1/Vmax
40

Km/Vmax

[S]/v
20

0

-Km

-2

(9)
(10)

0

[S]

2

4

Figure 3. Hofstee plot

(7)

Eadie plot: v/[S] vs v
𝑣𝑣
𝑣𝑣 = −𝐾𝐾𝑚𝑚 ×+ 𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚(8)[𝑆𝑆]
Plot v versus v/[S] (Figure 4) based on the modified version of
the Michaelis-Menten equation shown above. Determine theslope (a) and the intercept (b) of the straight line using the leastsquares method and then calculate Vmax and Km using equations(9) and (10), respectively. This plot is effective at wide range ofsubstrate concentration.
𝑉𝑉𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑏𝑏

60

(5)

0.09
0.08

Vmax
Slope = -Km

0.06
v

0.04

Vmax /Km

0.02

0

0

0.02

0.04
v/[S]

0.06

0.08

Figure 4. Eadie plot
17

LIFE SCIENCE APPLICATIONS

Measurement of trypsin activity
using fluorescence peptide substrate
Proteases are responsible for breaking down biological molecules into smaller polypeptide chains throughhydrolysis. Upon hydrolysis of the substrate (methylcoumarin-amide [MCA]), 7-amido-4-methylcoumarin (AMC)
and a water-bound trypsin peptide are produced. Trypsin is a protease that is commonly used in assays todetermine the enzymatic activity of a molecule. After cleavage of the substrate via hydrolysis, the trypsin
activity can be measured by monitoring the fluorescence intensity at 440 nm of the isolated product, AMC as
a function of time. This section shows the example of trypsin activity evaluation by fluorescence peptide MCA
substrate.
Boc-Gln-Ala-Arg-NH

H2N

O

O

+H2O

+ Boc-Gln-Ala-Arg-OH
CH3

CH3
Boc-Gln-Ala-Arg-MCA

Isolation AMC

Figure 1. The hydrolysis reaction of MCA by protease[Samples]- Intensity standardization sample:
50 μmol/L AMC solution- Enzyme solution:
10 nmol/L Trypsin bovine pancreas typeVIII
50 mmol/L Tris-HCl
0.15 mol/L NaCl
1.0 mmol/L CaCl2
0.1 mg/mL BSA- Substrate solution: Boc-Gln-Ala-Arg-MCA solution(Last concentration: 0.5, 1, 2.5, 5, 10, 20, 40 µmol/L)

Concentration foradjustment (μmol/L)

Last concentration(μmol/L)

240

40.0

120

20.0

60

10.0

30

5.0

15

2.5

6

1.0

3

0.5

[Measurements]
1) Fluorescence-spectrum measurement of AMC
Excitation and the Fluorescence spectrum of 50 µmol/L AMC were measured (Figure 2). It turns out that the
maximum emission wavelength from this result is 440 nm.
9000

6000
Int.
4000

Excitation
spectrum
Fluorescence
spectrum

2000

0
300

400

500

600

Wavelength (nm)Figure 2. Excitation/Fluorescence spectra of AMC
18[Measurement parameters]
Excitation wavelength:		
360 nm			(fluorescence spectrum)
Emission wavelength: 		
440 nm			(excitation spectrum)
Bandwidth (excitation):
5 nm
Bandwidth (emission):		
10 nm
Scanning speed:		
500 nm/min.
Data interval: 			
1 nm
Response: 			0.5 sec.
Sensitivity:			200 V

App. Note: 210-FP-0011

2) Vertical axis switching (into concentration from fluorescence intensity)
Intensity standardization was performed in order to change the vertical axis into the numerical value equivalent
to concentration.
50 µmol/L AMC solution of 0.5 mL is dropped at enzyme solution 2.5 mL. Fluorescence intensity of lastconcentration of 8.333 µmol/L AMC solution was set to 8333 µmol/L AMC solution.
3) Enzyme activity measurement
Substrate solution 0.5 mL of each concentration was dropped at enzyme solution 2.5 mL, and time course
measurement of the fluorescence intensity of the isolation AMC was performed to it. The results are shownin Figure 3.
30000

Last concentration
0.5 µmol/L
1.0 µmol/L
2.5 µmol/L
5.0 µmol/L
10.0 µmol/L
20.0 µmol/L
40.0 µmol/L

20000

Int.
10000

0

0

50

100

150

[Measurement parameters]
Excitation wavelength:
Emission wavelength:
Bandwidth (excitation):
Bandwidth (emission):
Data interval: 		Response: 			
Sensitivity: 			

360 nm
440 nm5 nm
10 nm
0.1 sec.
0.1 sec.
200 V

180

Time (sec.)

Figure 3 Monitoring results of an enzyme reaction
process[Analysis]
Each initial velocity was calculated from inclination of the time variation data of each substrate concentration,and the Lineweaver-Burk plot was performed (Figure 4). Km=5.99 and Vmax=35270 nmol/L·min-1 were obtained
from this result.
0.0004[Calculation results]
Calculation method: Lineweaver-Burk Plot35270
Vmax:		Km:		 5.99
1/v = 0.000170·1/[S] + 0.0000284

0.00031/v

0.00020.0001

0
-0.2

0

1/[S]

1

2

Figure 4. Lineweaver-Burk plot
19

LIFE SCIENCE APPLICATIONS

Reaction process monitoring byfluorescence stopped-flow system
Stopped-flow is the technique for monitoring the fast chemical reaction on millisecond time scale. After mixing
more than two solutions quickly, it monitors the reaction process through spectroscopies (fluorescence, circulardichroism or absorption). The measurement examples by using stopped-flow system are as below.
Synthesis of organic/inorganic compound- Property evaluation of novel metal complex and organic compound (porphyrins, fullerenes etc.)- Reaction process analysis of active oxygen, free radical, and anti-oxidative substance- Drug development, polymerization reaction
Biochemistry- Analysis of enzyme catalyst reaction and folding reaction process of protein- Analysis of interaction between ligand and protein/DNA- Reaction evaluation of artificial heme protein

Refolding

Unfolding
Denatured state

Native state

JASCO's stopped-flow system gives the time course data with high accuracy, and can calculate the reaction
rate by fitting the following reaction rate equation to the obtained data.
1-step reactionA𝑡𝑡
𝑌𝑌(𝑡𝑡) = 𝑎𝑎 ∙ 𝑒𝑒𝑒𝑒𝑒𝑒 (− )𝜏𝜏B

2-step reaction
A

B

C

Y(t): measured value
t: timeτ, τ1, τ2: time constantsa, b: constants

20

𝑌𝑌(𝑡𝑡) = 𝑎𝑎 ∙ 𝑒𝑒𝑒𝑒𝑒𝑒 (−

(1)

𝑡𝑡
𝑡𝑡
) + 𝑏𝑏 ∙ 𝑒𝑒𝑒𝑒𝑒𝑒 (− )
𝜏𝜏1
𝜏𝜏2

(2)

App. Note: 210-FP-0007

Acid unfolding of horse cytochrome C measured with a fluorescence stopped-flow system
The fluorescence characteristics of the tryptophan residue in proteins will vary depending on the structures
surrounding the residue. This characteristic of cytochrome C is derived from the tryptophan in the residue
position 59. The natural state of this tryptophan residue is so close to the Heme iron residue that the fluorescenceis quenched by nonradiative energy transfer to the Heme iron. When Cytochrome C is denatured by an acid,the distance between the tryptophan and Heme iron changes and the fluorescence intensity enlarges.
This section introduces the measurement example of the change in fluorescence intensity by the aciddenaturation of Cytochrome C as measured by the JASCO stopped-flow measurement system.
As shown in figure 1, the fluorescence intensity has been changed extremely according to the acid denaturationof Cytochrome C. The calculated range was 35 to 5000 msec and a 2-step reaction mechanism was applied
for the calculation. The calculated results show an excellent fit to the experimental data, and the reaction rate
constants of first step and second step are 433 msec. and 3611 msec., respectively.
[Sample]- Syringe 1: 10mL, 0.5 mg/mL Cytochrome C- Syringe 2: 10mL, 0.1 N sulfuric acid

300

250

Int.

200

Measured150
120

Calculated

0

1000

2000

3000

4000

5000

Time (msec.)

Figure 1. Fluorescence intensity monitoring forthe denaturation of Cytochrome C[Measurement parameters]
Excitation wavelength:
Emission wavelength:
Bandwidth (excitation):
Bandwidth (emission):
Data interval:
Response:
Sensitivity:
Accumulation:
Flow time:
Mixing ratio:
Flow volume:
Start data acquisition:

280 nm
340 nm
5 nm
5 nm
5 msec.
2 sec.
Manual4
35 msec.
S1 : S2 = 1 : 1
S1, S2: 100 μL
35 msec. before flowtime ends

Calculation range:
35–5000 msec.
Reaction rate formula: Y(t) = -142.6 × exp(-t/432.8) + -47.7112 × exp(-t/3611)
Step 1 time constant: 432.8 msec.
Step 1 rate constant: 0.002310 msec.-1
Step 2 time constant: 3611 msec.
Step 2 rate constant: 0.0002769 msec.-1
21

LIFE SCIENCE APPLICATIONS

Quantitative analysis of λDNA
using One drop accessory
One drop attachment is a microsampling accessory that enables fluorescence measurements to be obtainedwith only 5 µL of sample. It is the most suitable and effective for quantitative analysis of multiple samples suchas fluorescently-labeled DNA and various kinds of fluorescent dye.
1) Drop a sample on the cell4) Wipe the sample off2) Set the cover glass

3) Push the start button
This section demonstrates the use of the One Drop accessory to obtain fluorescence data and create acalibration curve of λDNA labeled with PicoGreen®.
[Results]
The fluorescence spectra of λDNA for a variety of concentrations is shown in Figure 1, and indicates that the
maximum emission wavelength is 523 nm.
9000
1000 ng/mL

8000

500 ng/mL
100 ng/mL
50 ng/mL

6000

10 ng/mLInt.

5 ng/mL

4000

1 ng/mL

2000

0
-1000

510

550

600
Wavelength (nm)650

Figure 1. Fluorescence spectra of λDNA labeled with PicoGreen®
22

App. Note: 260-FP-0010

The fluorescence intensity at the maximum emission wavelength (523 nm) was then measured five times foreach sample to ensure measurement reproducibility. Table 1 shows these measurement reproducibility resultsfor each sample concentration.
Table 1. Measurement reproducibility results
Conc. (ng/mL)
0

1

2

3

4

5

53.3

52.4

55.9

53.1

55.2

Ave.

S.D.

C.V (%)

54.0

1.49

2.8

1

63.6

68.1

65.9

66.5

65.1

65.8

1.68

2.6

5

110.3

106.7

105.1

104.0

110.0

107.2

2.86

2.7

10

157.6

155.5

156.1

153.0

151.7

154.8

2.39

1.5

50

447.1

465.3

460.2

455.8

469.0

459.5

8.56

1.9

100

865.9

856.9

848.3

850.6

853.9

855.1

6.86

0.8

500

3842.73831.03858.03828.93811.03834.3

17.42

0.5

1000

7766.27992.17925.37972.87805.77892.4101.15

1.3[Measurement parameters]
Measurement mode:
Excitation wavelength:
Bandwidth (excitation):
Response:

Emission
480 nm
20 nm
1 sec.

Emssion wavelength: 523 nm
Bandwidth (emission): 20 nm
Sensitivity:
620 V

A calibration curve was then created using the average value for each concentration from the reproducibility
results in Table 1 and is shown in Figure 2. The correlation coefficient is 0.9999 and the standard error is 6.819,
indicating a good fit.
8000

6000

Int. 4000

2000

0

0

200

400

600

800

1000

Concentration (ng/mL)

Figure 2. Calibration curve of λDNA labeled with PicoGreen®
Calibration curve equation :Correlation coefficient :Standard error :Int.= 7.780 × Conc.+ 57.5211
0.99996.819
23

MATERIAL SCIENCE APPLICATIONS

Fluorescence quantum yieldmeasurement
Fluorescence quantum yield is defined as the ratio of the number of photons emitted from sample as
fluorescence to the number of photons in the excitation light absorbed. Absolute method and relative method
are known as measuring methods.
Relative method
Relative method can calculate quantum yield of unknown sample by comparing the intensity of standard
fluorescence with unknown sample, and accordingly, the relative method is easier to get the results of quantum
yield.
[Calculation method]
In order to calculate relative quantum yield, the following parameters are required.
(1)Quantum yield of standard sample: Φst(2)Absorbances at excitation wavelength for the standard sample (st) and the unknown sample (x): Ast, Ax(3)Areas of fluorescence spectrum with spectral correction for the standard sample (st) and the unknown
sample (x): Fst, Fx(4)Average refractive index values in the wavelength range to calculate the area of fluorescence spectrum forthe standard sample (st) and the unknown sample (x): nst, nx*) Its information is required when the solvent of the standard sample is different from that of unknown
sample(5)Dilution ratio for the standard sample (st) and the unknown sample (x) respectively: Dst, Dx*) Its information is required when the standard or unknown samples for fluorescence spectrum measurement
are diluted
By using the parameters above, relative quantum yield of unknown sample, Φx is shown by the following
equation.

∅𝑥𝑥 = ∅𝑠𝑠𝑠𝑠 ∙ (24

𝐴𝐴𝑠𝑠𝑠𝑠𝐹𝐹𝑥𝑥
𝑛𝑛𝑥𝑥 2𝐷𝐷𝑥𝑥) ∙ ( ) ∙ ( 2) ∙ ( )𝐴𝐴𝑥𝑥
𝐹𝐹𝑠𝑠𝑠𝑠𝑛𝑛𝑠𝑠𝑠𝑠𝐷𝐷𝑠𝑠𝑠𝑠(1)

Absolute method
The absolute method allows to detect all the fluorescence from the sample and integrates using integrating
sphere. Therefore, its method enables more accurate quantum yield measurement.
[Measurement method](1)Measuring incident light
Set the cell with solvent on the sample cell holder in integrating sphere, and measure spectrum of incident
light (In case of the solid sample, confirm nothing is set on the sample cell holder in the integrating sphere,
and measure spectrum of the incident light). Obtained peak area is defined as area from incident light, S0(equivalent number of photons in the incident light).
(2)Measuring sample
Set the sample on the sample holder, and measure scattering and fluorescence spectra of the sample. Obtained
excitation wavelength peak area is defined as area scattered from sample, S1 (equivalent number of photons
which were not absorbed), and peak area in the emission wavelength range is defined as area emitted fromsample, S2.
Excitation light

Excitation light

To detection

To detection

Fluorescencet

Sample holder

Sample holder

Measuring incident light

Measuring sample(3) Calculating quantum yield
Calculate in accordance with the following.
•

Sample absorption (%) = (S0 − S1)/S0 × 100•

External quantum yield (%) = S2/S0 × 100•

Internal quantum yield (%) = S2/(S0 − S1 ) × 100

S0: Area of incident lightInt.

S1: Area of scattered light of sample= Number of photons not absorbed

S2: Area of luminescence

Wavelength (nm)JASCO can provide the fluorescence quantum yield measurement system (relative method or absolute
method), and also propose the proper solution to meet the user's requirement.
25

MATERIAL SCIENCE APPLICATIONSInternal quantum efficiency
measurement of phosphor powders
Some white LEDs consist of two types of materials: a luminescent diode that emits a blue light in thenear-UV region, and a phosphor that emits visible light from the absorption to the near-UV region.
Determining a phosphor’s internal quantum efficiency is an important parameter in evaluating theemission efficiency of a white LED, and the standards about its evaluation method (JIS R1697/ISO 20351)
have been published. This section illustrates the measurement reproducibility of the internal quantum
efficiency of two phosphor powders from the evaluation of a white LED by using the dedicated system
which is compliant with JIS R 1697 /ISO 20351.
[Measurement procedure]
1) Fill BaSO4 into the cell, and measure the incident light spectrum.

Excitation light

BaSO4

Integrating sphere

Excitation light2) Fill the sample into the cell, and measure the spectrum that includes excitation/scattering light andfluorescence.

Excitation light

SampleExcitation light
Integrating sphere

Fluorescence3) Perform the spectrum correction by using calibrated light source, and apply it to measured spectra.
[Samples]- Yellow phosphor, red phosphor[Measurement parameters]
Excitation wavelength:
Measurement range:
Bandwidth (excitation):
Bandwidth (emission):
Scanning speed: 		Response: 			
Sensitivity: 			
26

455 nm380–780 nm
5 nm
5 nm
500 nm/min.
0.1 sec.
355 V

App. Note: 190-FP-0017[Results]
To confirm reproducibility, five repeat measurements were made for each sample (by refilling the sample cell).
High reproducibility was obtained for both phosphor measurements as indicated by the difference between
the maximum and minimum values of the internal quantum efficiency (within 1.5% with a coefficient of varianceof 0.6%).
6000

400

Incident light spectrum
Fluorescence spectrum (#1)
Fluorescence spectrum (#2)

300

Fluorescence spectrum (#3)

4000

Fluorescence spectrum (#4)
Fluorescence spectrum (#5)

Int.

Int.

200

2000
100

0
380

500

700

600
Wavelength (nm)

0
465

780

500

700

600
Wavelength (nm)780

Figure 1. Fluorescence spectra of yellow phosphor
Table 1. Calculated results of internal quantum efficiency (yellow phosphor)
1

2

3

4

5

Ave.

S.D.

C.V.

Light volume absorbed by sample

87.6 %

87.4 %

87.3 %

87.2 %

87.1 %

87.3 %

0.19

0.22

External quantum efficiency

80.7 %

80.3 %

79.9 %

79.7 %

79.0 %

79.9 %

0.64

0.80Internal quantum efficiency

92.1 %

91.9 %

91.6 %

91.4 %

90.7 %

91.5 %

0.54

0.59

6000

300
Incident light spectrum
Fluorescence spectrum (#1)
Fluorescence spectrum (#2)

4000

Fluorescence spectrum (#3)

200

Fluorescence spectrum (#4)
Int.

Fluorescence spectrum (#5)

Int.

2000

100

0
380

500

700

600
Wavelength (nm)

0
500

780

700

600

780

Wavelength (nm)Figure 2. Fluorescence spectra of red phosphor
Table 2. Calculated results of internal quantum efficiency (red phosphor)
1

2

3

4

5

Ave.

S.D.

C.V.

Light volume absorbed by sample

78.2 %

77.6 %

77.9 %

77.8 %

77.9 %

77.9 %

0.22

0.28

External quantum efficiency

65.8 %

64.9 %

65.9 %

65.4 %

66.1 %

65.6 %

0.48

0.73

Internal quantum efficiency

84.1 %

83.6 %

84.6 %

84.1 %

84.9 %

84.3 %

0.50

0.60

27

MATERIAL SCIENCE APPLICATIONS

Phosphorescence measurement

Light emission as a result of photo-excitation can occur
partially in the form of fluorescence and partially as
phosphorescence. Phosphorescence occurs when
an intersystem crossing occurs, and electrons cannot
return directly to the ground state, but instead makea transition to an intermediate state. They then relax
slowly to the ground state, resulting in phosphorescence.
Fluorescence occurs on a timescale of nanoseconds,
whereas phosphorescence can continue for much longer
periods. When electronic transitions occur in a substance,electron pairs generally have opposite spins (Pauli’s
exclusion principle). However, when an intersystemcrossing occurs, the spins are the same. Since this is aforbidden state, relaxation to the ground state is slow.
When measuring phosphorescence substance by
spectrofluorometer, the sample is irradiated with excitation
light, and then the light after cutting the excitation lightshould be observed.

Shutter

Open

Close

Open

Signalintensity

Phosphorescence
Fluorescence

Fluorescence

Phosphorescence

Phosphorescence

Measurement time

Figure 2. Observed signal when opening/closing
the shutter
Phosphorescence quantum yield measurement

Singletexcited stateintersystemcrossing

Absorbance

Tripletexcited state

10 -15 sec.
Fluorescence
10 -6 –10 -9 sec.

Phosphorescence
10 -3 –1 sec.
Ground state

Figure 1. Phosphorescence due to intersystemcrossing
Since fluorescence and phosphorescence are mixed
while the sample is irradiated with excitation light, thephosphorescence only cannot be observed. Therefore,
utilizing the property that lifetime of phosphorescence islonger than the one of fluorescence, cutting the excitation
light enables to observe phosphorescence only.
JASCO's spectrofluorometer has the shutter at the
monochromator of excitation side, which enables tomeasure the phosphorescence. Figure 2 shows thesignal of fluorescence/phosphorescence when opening/closing the shutter. When opening the shutter, the sample
is irradiated with excitation light, and the observed
signal is mixture of fluorescence and phosphorescence.
When closing the shutter, the phosphorescence only isobserved.
This section shows the evaluation examples ofphosphorescence.

Phosphorescent compounds are frequently used as the luminescent layer in organic electro-luminescentdevices. Quantum yield measurement of phosphorescence substances is required for developing suchmaterials. JASCO developed a new dedicated system for calculating quantum yield from the measured
phosphorescence spectra at 77K. A dedicated system was evaluated by using benzophenone which is a
representative phosphorescence substance. Figure 3 illustrates the measured spectra and Table 1 shows
the calculation results of quantum yield (φ). The calculated phosphorescence quantum yield was 0.93 that
corresponds well with the literature-based value of 0.9[1].
[1] The chemical society of Japan, Courses in Experimental Chemistry 3 basic physical chemistry, Maruzen ISBN: 4-621-07303-6
28

App. Note: 190-FP-0006, 250-FP-0023

600
Incident spectrum

Table 1. Phosphorescence quantum yield ofbenzophenone

Sample spectrum
Sample spectrum (× 25)
400

Int.

Int.

200

0
300

400

600

500
Wavelength (nm)Incident photon number: S04954.3

Photon number unabsorbed by sample: S1

4074.0

Photon number emitted by sample: S2

819.3

Quantum yield [Measured]

0.93

Quantum yield [Literature]

0.90

650

Figure 3. Spectra of benzophenone
Detection of Phosphorescence from Singlet Oxygen using NIR Spectrofluorometer
Singlet oxygen is highly active and destroys biological molecules. Recently, its use for destroying cancer cells
or bacteria has been researched. Herein, this section shows measurements of phosphorescence at 1270 nm
emitted by singlet oxygen using a near-infrared spectrofluorometer with Eosin Y as a photosensitizer.
The excitation-emission matrix measurement of singlet oxygen reveals a suitable excitation wavelength aswell as whether the singlet oxygen has been generated. Near-infrared spectrofluorometer is effective tool for
research of singlet oxygen.
700

2

0.8

Excitation wavelength (nm)

600
0.6

Int. 0.4
500
0.2

0
1200

400

300
1350
1200
1250
1300
Emission wavelength (nm)

1250
1300
Wavelength (nm)

1350

0

Figure 4. Excitation-emission matrix and fluorescence spectra (excitation wavelength: 525 nm) of
singlet oxygen.
Acknowledgment
Special thanks to associate professor Takunori Harada (Department of Integrated Science and Technology, Faculty of
Science and Technology, Division of Applied Chemistry, Oita University) who provided the sample.
29

MATERIAL SCIENCE APPLICATIONS

Remote fluorescence measurement
using optical fiber
JASCO has various accessories for supporting the user's measurement, and these accessories can bemounted to spectrofluorometer easily. One of JASCO's accessories is optical fiber interface, which enables thefluorescence measurement by holding the probe close to or immersed in the sample. A spectrofluorometerwith optical fiber can be used for a variety of measurements, such as when the sample is larger than thesample chamber, to follow an in-situ reaction, or for measurement in hazardous environments, such as high/low
temperature and high pressure. This section shows the measurement example by using spectrofluorometerwith optical fiber.
[Measurement]
Fluorescence measurements of fluorescent reflector (red/yellow) were performed by using spectrofluorometer,
optical fiber and dark chamber. The spectral correction was also performed by using the reflected light of thecalibrated WI lamp from standard white plate.
Sample measurement

Calibrated light source measurement
Spectrofluorometer

Spectrofluorometer

Calibrated
light source
Ex.

Standard
white plateEm.
Branched
optical fiber

Branched
optical fiber

Figure 1. Overview of measurement system
Figures 2 shows the measurement results. The profiles of the spectra of each colored sample measured using
both the optical fiber and film holder were consistent with each other.
This system is effective in the case such as the measurement of a sample which is larger than sample chamber
and the measurement under the severe environment.
100

100
Optical fiber

8080Film holder

60

Optical fiber
Film holder

60

A.U.

A.U.
40

40

20

20

0
400

500

600
Wavelength (nm)

700

800

0
400

500

600
Wavelength (nm)

700

800

Figure 2. Peak normalized fluorescence spectrum of the yellow fluorescent reflector (left) and the red
fluorescent reflector (right), excitation wavelength: 310 nm30

App. Note: 260-FP-0018

The solution examples by using optical fiber are shown as below.
Spectrofluorometer

Spectrofluorometer
Light emitting
element

Light emitting
element

Integrating
sphereLamp evaluation (oriented type)

Lamp evaluation (Omnidirectional type)
Spectrofluoromenter

Spectrofluorometer
SampleOptical fiber
Optical fiber
SampleAutostage

In-situ measurement

Mapping measurement

In addition, JASCO can provide the useful optional
software to convert the luminous/rendering color into
numerical values and to evaluate them quantitatively.
It has the calculation function of each color model
which CIE (International Commission on Illumination)
has published, and is also compliant with JIS
regulation (color specification). Since it includes pass/
fail evaluation function, it will be powerful tool tomanage the luminous color of product.

[Luminous Color Analysis] program
31

MATERIAL SCIENCE APPLICATIONS

Evaluation of up-conversion phosphor

Generally, fluorescence appears on the long wavelength side, which means that its energy is lower than theone of excitation light (the light absorbed by the sample). On the other hand, in the case of up-converting
phosphor, the energy of fluorescence is higher than the one of excitation light because two or more photons
involves its reaction, and the fluorescence appears on the short wavelength side. This is particularly usefulfor evaluating labels used for biological imaging, materials in infrared solar cells and development of inks foranti-counterfeiting.
JASCO has developed the up-conversion measurement system, which can detect very small fluorescence byusing laser. This section shows the measurement examples of the up-conversion phosphors.
<Conventional fluorescence><Up-conversion fluorescence>
Thermal relaxation
Secondexcited state
Absorption

Thermal relaxation
First excitedstateFirst excitedstate
Fluorescence

Absorption

Ground state

Absorption

Fluorescence

Ground state

Mechanism of up-conversion fluorescence
10000

8000

6000
Int.
4000

2000

0
200

The fluorescence at visible light region could
be observed when irradiating 980 nm lightwhich the human’s eye cannot observe.
32

400

600
Wavelength (nm)

800

900

Our up-conversion fluorescence measurement
system with 980 nm near-IR laser could
observe the up-conversion fluorescence.

App. Note: 190-FP-0014, 190-FP-0015

Up-conversion quantum yield measurement of phosphors with heavy rare earth element[Samples]- YTa7O19: Er10, Yb40
[Measurement parameters]
Excitation wavelength:
980 nmBandwidth (emission):
5 nm
Scanning speed:		
1000 nm/min.
Data interval:		
0.2 nmResponse:			0.2 sec.
Laser output:		
150 mW
Each sample was measured three times to evaluate the measurement reproducibility. The three measurements
are overlaid and shown in different colors in Figures 1. Table 1 shows the quantum yield measurement result.
This result demonstrates that the up-conversion system can evaluate quantum yields at levels of less than
1%. Since the quantum yield of up-conversion phosphor is generally very low, combining laser source with
spectrofluorometer enables to observe very weak fluorescence. This dedicated system is expected to beeffective tools for studying the excitation process of up-conversion phosphor.
60

500000400000

Excitation light

300000

Scattered light

40
Int.

Int.
20000020

100000
0

0
950

960

980

1000 1010

450

Wavelength (nm)

600

700

800

870

Wavelength (nm)Figure 1. The scattered light spectra and the fluorescence spectra(Blue: first, Green: second, Red: third)
Table 1. Result of internal quantum yield

1

Area ofExcitation light

Area of
Scattered light

2.80E+06

2.14E+06

Area ofAbsorption
663270Area ofFluorescence

Sampleabsorbance (%)

Internal quantum
yield (%)

1453.86

23.69

0.22

2

2.73E+06

2.14E+06

5878401378.84

21.57

0.23

3

2.83E+06

2.14E+06

6876201433.80

24.28

0.21

Acknowledgment
Data courtesy of Dr. Kouji Tomita, Department of Chemistry, School of Science, Tokai University
33

ENVIRONMENTAL SCIENCE APPLICATIONS

Evaluation of environmental waterusing PARAFAC
Recently, excitation-emission matrix (EEM) measurements and parallel factor (PARAFAC) analyses have been
widely used for evaluating the quality of environmental water, such as in rivers and lakes.
Such water often contains compounds such as humic acid, tryptophan, and artificial chemicals. Therefore,
multiple overlapping peaks appear in the EEM. PARAFAC is a very powerful analysis method in such situations
and can separate the principal components of EEM. It can be used to predict the compounds present bycomparing with published EEM. It can also be used for quantitative analysis.
In this section, a PARAFAC analysis of EEM for river water is described.
Upstream[Samples]
30 water samples (3 samples each at 10 locations shown in figure)Sampling point

1
2[Measurement parameters]
Excitation range:		
250–450 nm
Emission range:		
260–700 nm
Bandwidth (excitation):
5 nm
Bandwidth (emission):
5 nm
Excitation step:		
5 nm
Scanning speed:		
1000 nm/min.
Response:			50 msec.
Sensitivity:			Medium

3

4
5
6

Sewagetreatment
plant

7
8
9
10
Downstream

Figure 1 Schematic diagram ofsampling points

450
400
350
300
250
260

400

500

600

700

Excitation wavelength (nm)

Excitation wavelength (nm)[Results]
Representative EEM for each sampling point are shown in figure 2.
450
400
350
300
250
260

Emission wavelength (nm)

450
400
350
300
400

500

600

Emission wavelength (nm)Point 3

34

500

600

700

Point 2 (downstream of sewage plant)

700

Excitation wavelength (nm)

Excitation wavelength (nm)Point 1 (upstream of sewage plant)

250
260

400

Emission wavelength (nm)

450
400
350
300
250
260

400

500

600

Emission wavelength (nm)Figure 2. EEM for each sampling point

Point 4700

Based on the literature , components 1 and3 appear to be humic acid-like compounds
and component 2 appears to be tryptophan.
Component 4 is a residual error. Score plots forthe different components are shown below.

450

450

400

400

Excitation wavelength (nm)A PARAFAC analysis was applied to 30 sets ofEEM. When the number of components was set to4, the results of PARAFAC analysis were as shownin figure 3.

Excitation wavelength (nm)App. Note: 260-FP-0025

350

300

350

300

[1]

300

400
500
600
Emission wavelength (nm)

250

700

300

Component 1
450

450

400

400

350

300

250

300

400
500
600
Emission wavelength (nm)

400
500
600
Emission wavelength (nm)

700

400
500
600
Emission wavelength (nm)700

Component 2

Excitation wavelength (nm)

Excitation wavelength (nm)[1] Chen. W. et.al. Environ. Sci. Tech. 2003, 37,
5701-5710.

250

700

350

300

250

300

Component 3

Component 4

Figure 3. Components determined by PARAFAC(assuming 4 components)
Clustering based on the sampling points can be seen. The results indicate a sudden increase (about 2.5
to 3 times) in the concentration of humic acid-like compounds and tryptophan downstream of the sewage
treatment plant, but then no rapid increase between points 2 and 5.
110

100

100

90

80
70
60
50
40

Point 3
Point 4607080
Scores on Comp 1

90

Component 1 vs. Component 2

100

110

Point 8
Point 9Point 10

40

20
50

Point 750

20
40

Point 6

60

30

30

Point 5

70

30

10
20

Point 280
Scores on Comp 3

Scores on Comp 2

90

Point 1

10
10

20

30

40

50
60
70
80
Scores on Comp 2

90

100

110

Component 2 vs. Component 3

Figure 4. Score plots

Acknowledgment
Special thanks to Prof. K. Sankoda (Saitama University) who provided the sample.
35

ENVIRONMENTAL SCIENCE APPLICATIONSHigh-Speed Excitation Emission Matrix
Measurement of Olive Oil
Recently, Excitation-Emission Matrix (EEM) is drawing attention as the method for determining the type, quality,and geographical origin of food products. To firmly establish EEM as a reliable method for this type of analysis,
more references are required, and therefore many more samples should be measured (from hundreds tothousands).
In order to increase sample throughput, a system has been developed for high-speed EEM measurement
using the spectrofluorometer coupled with a multi-channel spectral detector. To demonstrate the ease-of-useof this high-speed measurement system, this application note reports the results for the measurement of avariety of olive oil samples. In addition, this note shows the use of EEM interpretation for characterizing theolive oil samples.
[Samples]Solutions of extra virgin (A, B, C, D, E, and F) and pure (G and H) olive oil were diluted in hexane at a 1:350 ratio
to obtain an absorbance less than 0.02 A.U.
[Results]
EEM after processing are shown in Figure 1. The peak locations in Figure 1 and literature* were used to estimate
the components of the samples and are the following: oxidation product (Excitation: 300 to 400 nm, Emission:
320 to 500 nm) and chlorophyll (Excitation: 300 to 700 nm, Emission: 650 to 800 nm).
*Kongbonga YGM, Ghalila H, Onana MB, Majdi Y, Lakhdar ZB, Mezlini H and Ghalila SS, Food and Nutrition
Sciences, 2, 692-699, (2011).
10000

500
400
300
320 400

500

600

700

500
400
300
320 400

800

500

600

700

600
500
400
300
320 400

800

500

600

700

600
500
400
300
320 400

800

500

600

700

Emission wavelength (nm)Emission wavelength (nm)Emission wavelength (nm)Emission wavelength (nm)a) EXV olive oil Ab) EXV olive oil Bc) EXV olive oil Cd) EXV olive oil D

500
400
300
320 400

500

600

700

800

600
500
400
300
320 400

500

600

700

800

600

0
10000

500
400
300
320 400

500

600

700

800

600
500
400
300
320 400

500

600

700

Emission wavelength (nm)Emission wavelength (nm)Emission wavelength (nm)Emission wavelength (nm)e) EXV olive oil Ef) EXV olive oil Fg) Pure olive oil Gh) Pure olive oil H

Figure 1. EEM of olive oil samples

36

800

700
Excitatinoo wavelength (nm)

600

700
Excitation wavelength (nm)700
Excitation wavelength (nm)700
Excitation wavelength (nm)

600

700
Exitation wavelength (nm)

600

700
Excitation wavelength (nm)700
Excitation wavelength (nm)

Excitation wavelength (nm)

700

800

0

App. Note: 080-FP-0021

Parallel factor analysis (PARAFAC) was performed on the EEM and the number of component spectra was set
to 2. Figure 2 shows the component EEM calculated by PARAFAC. The first component EEM is chlorophyll andthe second is an oxidation product.
700

700
650
Excitation wavelength (nm)

Excitation wavelength (nm)

650
600
550
500
450
400
350
300

600
550
500
450
400
350

400

500
600
700
Emission wavelength (nm)

300

800

400

500
600
700
Emission wavelength (nm)800

Figure 2. First (left) and second (right) component EEM calculated by
PARAFAC.
Figure 3 shows the score plot for the first component (chlorophyll) and the second component (an oxidation
product). Regarding the distribution in the extra virgin olive oil, the score for the first component is large while
the second is small. However, the distribution in the pure olive oil shows the score of first component is small
while the second is large. This trend is related to the production process of the olive oil. Extra virgin olive oil
is produced by squeezing and filtering the fruit of the olive without any chemical treatment process, and theacidity of the olive oil is less than 0.8%. On the other hand, pure olive oil is a blend of virgin olive oil and refinedolive oil, so the acidity of pure olive oil is less than 1%. In addition, the chlorophyll content of pure olive oil issmall because it is reduced during the production process for refining the olive oil.
These results demonstrate JASCO’s high-speed fluorescence measurement system and the use of PARAFAC
analysis for the interpretation of EEM. PARAFAC was used to extract the component EEM to estimate thecompounds from the component EEM. The score plot calculated by PARAFAC can provide information about
the characteristics of a sample and is useful for determining sample type and quality.
6

x 10 4

EXV olive oil A
EXV olive oil B

Second component

5

EXV olive oil C

Pure olive oil

EXV olive oil D
EXV olive oil E

4

EXV olive oil F
Pure olive oil G

3

Pure olive oil H

2

EXV olive oil

1
0

0

0.5

1

1.5
First component

2

2.5

3
x 105

Figure 3. Score plot of the first and second
components analyzed by PARAFAC
37

ENVIRONMENTAL SCIENCE APPLICATIONSPetroleum product assessment bycounmarin analysis
To prevent the production of illegal light diesel oil, which contains kerosene or heavy oil, 1 ppm of coumarinis added to either the kerosene or a heavy oil as a discriminator. The analysis procedure for determining
the discriminator and its mixing concentration is standardized by the Japan Petroleum Institute and uses aspectrofluorometer to determine concentration (JPI-5S-71-2010, Petroleum products - Determination of
coumarin - Fluorescence spectrometry). Coumarin analysis method is as below;- Conventional method (B method); usage of reagents is large, and separatory funnel is required for preparation.
- Simplified quantitative method (A method); flat bottom test tube with screw cap is used, and usage of reagentsis small.
- Simplified qualitative method: fluorescence colorimeter is used for its method, which examines whether light
diesel oil includes coumarin additive oil.
JASCO can provide dedicated coumarin measurement system, which can determine the coumarin concentration
without feeling the stress. This section shows the measurement procedure of A method by using dedicatedcoumarin measurement system[Principle]
Coumarin is hydrolyzed in alkaline solution and becomes cis-O-hydroxycinnamic acid. The cis-O-hydroxycinnamic
acid is then isomerized by ultraviolet radiation and converted to trans-O- hydroxycinnamic acid. Since trans-Ohydroxycinnamic acid fluoresces, the product can be quantified using fluorescence spectroscopy.
H
Hydrolysis

O
Coumarin

O

C

C

H

H
COOH

PhotoisomerizationOH
Cis-o-hydroxycinnamic acid

C

C

COOH
H

OH
Trans-o-hydroxycinnamic acid

Figure 1. Hydrolysis and photoisomerization of coumarin.
[Sample preparation](1) Alkaline solution
Adds 10 g of sodium hydroxide and 20 g of sodium nitrate to 100 mL volumetric flask, fills ultrapure
water to the calibration line. After dissolving, stores its solution in a polyethylene vessel.
(2) Alcohol solution
Mixes 80 mL of 1-butanol and 60 mL of ethanol.
(3) Coumarin standard stock solution (10,000 mg/L)
Adds 1 g of coumarin to 100 mL volumetric flask, and fills toluene to the calibration line.
(4) Coumarin standard solution (100 mg/L)
Adds 5 mL of coumarin standard stock solution to 500 mL volumetric flask, and fills n-dodecane tothe calibration line.
(5) Coumarin standard solution (1 mg/L)
Adds 5 mL of coumarin standard solution (100 mg/L) to 500 mL volumetric flask, and fills n-dodecaneto the calibration line.
38

App. Note: 050-FP-0024[Measurement procedure]
The standard samples are mixed in test tubes, according to the specified ratios in Table 1. Shaking the samples,the coumarin is extracted in the alkaline solution, and hydrolyzed. The photoisomerization reaction is inducedby irradiating the sample using a spectrofluorometer and an excitation wavelength of 360 nm. The fluorescenceintensity is detected at 500 nm and used to generate a calibration curve.
Table 1. Mixing ratios of the standard solution
Additive
Concentration(%)

Coumarin
Solution(1 mg/L) (mL)n-dodecane(mL)

Alkaline
Solution (mL)

Alcohol
Solution (mL)

0.0

0.0

7.0

5.0

8.0

10.0

0.1

6.9

5.0

8.0

40.0

0.4

6.6

5.0

8.0

80.0

0.8

6.2

5.0

8.0

120.0

1.2

5.8

5.0

8.0

(1) Adds the sample, n-dodecane, alkaline solution, alcohol solution and stirrer bar to the test tube.
(2) Shakes the sample by the stirrer (240 r.p.m., 3 minutes). The coumarin is extracted in the alkaline solution,and hydrolyzed.
(3) After shaking, the sample is kept stationary for 5 minutes to allow for separation of the solution layers (top:light diesel oil, middle: alcohol, bottom; alkaline).
(4) Mounts the test tube to the spectrofluorometer. Stirring the bottom layer (alkaline solution), irradiates its
solution with UV light (360 nm) for 5 minutes so that the photoisomerization reaction is induced. Afterphotoisomerization, stops stirring, and measures the fluorescence intensity (excitation wavelength: 360 nm,
emission wavelength: 500 nm). And then, generates the calibration curve by using obtained values.
(5) When examining the unknown sample, adds 1 mL of unknown sample, 6 mL of n-dodecane, 5 mL of alkaline
solution and 8 mL of alcohol solution to the test tube. Performs the similar processing, and measures thefluorescence intensity (excitation wavelength: 360 nm, emission wavelength: 500 nm). Calculates thecoumarin mixing ratio by using calibration curve.

[Case of 80 %]Coumarin (1 mg/L): 0.8 mL
N-Dodecane: 6.2 mL
Alkaline solution: 5.0 mL
Alcohol solution: 8.0 mL

Shaking by stirrer(3 min.)Stationary (5 min.)

Irraduating thebottom layer withUV (360 nm), andthen measuring
fluorescence intensity

39

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

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.