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JASCO Applications BookSpectrophotometers

JASCO Corporation was founded in 1958 toprovide the scientific community with opticalspectroscopy products.

In the mid-1950's a group of researchers in theInstitute of Optics of what is now TsukubaUniversity needed an InfraredSpectrophotometer for their research.Since a commercially available instrument wasnot yet existing at the time, they undertook thechallenge to develop their own.

The result was quite a success - a reliableinstrument with excellent optical performance.As a second result, other research groups askedthem to replicate the instrument for use withintheir laboratories.

Company Presentation

“serving the Science and Technology World by providing most advanced analytical

instrumentation”

With the introduction of HPLC in the mid-1970'sJASCO's experience in highly sensitive and accurateoptical systems led to the development of a seriesof chromatographic detection systems. Fixed andvariable wavelength UV/Visible and Fluorescencedetectors were introduced featuring excellentsensitivity and reliability in compact modules. Inorder to offer complete HPLC systems JASCOdeveloped a variety of novel solvent deliverysystems as well as other accessories such as columnovens, autosamplers, and PC based control andanalysis software.

Today JASCO offers a wide variety of HPLC modules,accessories and analysis software. The new JASCOLC-4000 Liquid Chromatography series is designedto operate at pressures approaching 15,000 psi foreither gradient or isocratic separations, providingresearchers with a powerful tool when using thenew generation of small particle columns. LC-4000Series includes a versatile series of componentsoffering unique flexibility to build systems forroutine and specialized applications. LC-4000features the widest choice of optical HPLC detector:UV, diode array, fluorescence, chemiluminescence,CD, chiral and refractive index detector.

Finally JASCO’s modular Supercritical FluidChromatography and Supercritical Fluid Extractionplatforms provide a low-cost, fast, green technologywith reliable and worry-free performance for a widevariety of applications.

Over the years the JASCO product line has grownto cover instruments used, not only in researchbut also for routine analysis applications in areassuch as quality control, environmental analysis,and process control. The current spectroscopyproduct line encompasses instrumentation forthe following methods:

• UV/Visible and NIR• Microscope Spectrophotomers • FT-IR, microscope FT-IR and FT-Raman• Dispersive RAMAN• Polarimeters• Spectrofluorometers• Portable Raman• Portable FT-IR• Fully Automated Dissolution Tester

JASCO is also the world leader in the field of Circular Dichroism Spectropolarimeters andVibrational Circular Dichroism Spectrometers. JASCO has a strong global presence, supplying

customers in over 45 different countries.

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JASCO Europe is in charge for marketing, sales,service and support for all Jasco productsthroughout Europe, Middle East and Africa.

JASCO Europe

JASCO Europe S.r.l.Via Cadorna, 1 - 23894 Cremella (LC)

Tel. +39-0399215811Fax +39-0399215835

[email protected]

www.jasco-europe.com

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Make the most of your investment with

JASCO Service and Support

JASCO Service and Support agreement plans aredesigned for those laboratories pursuingsuperior productivity through the highest levelof professional services.The use of automated instrumentation is theright approach to meet today's laboratoriesproductivity requirements, reducing analysis runtimes, enhancing sample throughput, andincreasing analytical accuracy and precision. Inthis view, preventive maintenance is veryimportant to maximize laboratory uptime andavoid unexpected expenses.

In addition to the analytical goal, properinstallation and maintenance are required toachieve optimal performance. JASCO providesflexible service and support managementsolutions focused on your laboratory realobjectives.

With its service network, JASCO is ready to maintainthe perfect reliability of customer's instrumentationand minimize the laboratory down time.

· Superior productivity· Optimized analytical performance· Lower cost of ownership· Extended instrument life

If your laboratory has specific Service and Supportrequirements, JASCO can help you with customizedcontract agreements. In addition, a full set ofInstallation Qualification (IQ), OperationalQualification (OQ), and Performance Qualification(PQ) tests are available to verify the system properinstallation, operation and performance,respectively.

Get the most from your investment with

JASCO Training Courses

JASCO Training Courses ensure maximum skilldevelopment for the best value of your laboratory.Our team of highly-experienced specialists can helpyour staff to get the most from your instrumentreducing your analysis run time and improveperformance.

Build your knowledge with JASCO Training Courses:

· Instrument and Software operation· troubleshooting· Maintenance· Calibration· Applications and Methods developments· Operating Techniques

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The FT/IR-4000 and FT/IR-6000 models represent abroad range of instrumentation that redefineinfrared spectroscopy as a powerful yet easy to usetechnique in a compact and reliable line ofinstruments with the highest signal-to-noise ratio.

All models are controlled by Spectra Manager™ II ,JASCO’S powerful cross-platform spectroscopysoftware package with USB communication.

All models feature an auto-alignment functionwhich maintains instrument optical alignment afterbeamsplitter changes or instrument movement.

IRT-5100 – IRT-5200FT/IR Microscopes

V-730 – V-730bio – V-750 – V-760 UV-Vis Spectrophotometers

Spectroscopy Product Portfolio

V-770 – V-780UV-Vis/NIR Spectrophotometers

With more than fifty years of experience in thedesign of spectrophotometers, JASCO offers acomplete range of UV-Vis/NIR instruments.The V-700 series consists of six distinct modelsdesigned to meet a wide range of applicationrequirements.

From an innovative optical layout to a simplecomprehensive instrument control and dataanalysis software interface, the V-700 seriesdoes not compromise on accuracy, performanceor reliability.

All spectrophotometers are controlled bySpectra Manager™ II, JASCO’s powerful cross-platform spectroscopy software package withUSB communication.

FT/IR-4600 – FT/IR-4700FT/IR Spectrometers

FT/IR-6600 – FT/IR-6700 – FT/IR-6800FT/IR Spectrometers

IRT-7100 – IRT-7200FT/IR Microscopes

JASCO is proud to release four innovative FT-IRMicroscope, the IRT-5000 and IRT-7000, providingseveral new functions that drastically improveinfrared micro-spectroscopy analysis.

Both microscope systems can be easily interfacedwith either the FT/IR-4000 or FT/IR-6000spectrometer, offering the most advancedmicroscopy and imaging systems available in themarket today.

The microscope system automatically scans thespecified points or area, rapidly collecting a fullspectrum of each point without moving the samplestage.

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The performance expected on a micro-Ramanspectrometer are fully provided with the JASCONRS-5000/7000 series Raman systems, assuringconsistent performance for rapid acquisition of highquality data with automated system control andminimal optical adjustments.For application expansion, an automated multi-grating turret, 2 internally mounted detectors and amaximum of 8 lasers ranging from the UV throughthe NIR are capable of integration with theinstrument system.Spectra Manager™ II for the NRS-5000/7000 offersrevolutionary features to simplify previously difficultmeasurement and analysis tasks, while addingvarious user-support tools such as auto-fluorescence-correction, wavenumber correction,intensity correction, and a novel user-advicefunction.

MSV-5100 – MSV-5200 – MSV-5300UV-Vis/NIR Microscopes

NRS-4100 Laser Raman Spectrometer


The system offers space-saving, automatedswitching laser light source and alignmentadjustment to assist the analysis, NRS-4100 iseasily used to quality control as well as researchand development.

The micro-Raman NRS-4100 is equipped withmeasurement assist function that can be easilysetup operation and a user advice function thatautomatically analyzes the spectrum and obtaina high-quality data even at the first time.

The automatic XYZ stage is equipped with asample search function. Using a newlydeveloped algorithm (patent pending) themicroscope image, sample search function hasused to set the measurement positionautomatically and gives you data from thelocation that is automatically registered with theclick of a button measurement.

NRS-5100 – NRS-5200 Laser Raman Spectrometers

NRS-7100 – NRS-7200Laser Raman Spectrometers

The MSV-5000 series is a microscopicspectrophotometer system providingtransmittance/reflectance measurements of amicroscopic sample area with a wide wavelengthrange from ultraviolet to near infrared.

MSV-5100 Spectrophotometer is a dedicated UV-Vismicroscope with a wavelength range of (200-900nm).

MSV-5200 Spectrophotometer includes a Peltier-cooled PbS detector and has a wavelength range of(200-2700 nm).

MSV-5300 Spectrophotometer incorporates anInGaAs detector to obtain optimized NIRmeasurements and has a wavelength range of (200-1600 nm). 5

P-2000Digital Polarimeter

The P-2000 is designed as a customizablepolarimeter with various options for a range ofapplications and budgetary requirements.

Options such as polarizers, wavelength filters, lampsand photomultiplier detectors provide a wide rangeof analytical wavelengths from UV-Vis to NIR.

A newly redesigned intelligent remote module(iRM) with a color LCD touch screen convenientlyguides the operator through routines from dataacquisition to data processing. The obtained datacan be automatically printed to USB printers, orsaved to a compact flash memory card for furtherprocessing on a PC.

J-1100 – J-1500 – J-1700Circular Dichroism Spectropolarimeters


The latest effort in the JASCO commitment tolead the field of Circular Dichroism.

Unparalleled optical performance and optionallyavailable measurement modes are combined ina manner to make the J-1000 SeriesSpectropolarimeter, a true "chiro-opticalspectroscopy workbench“, able to work up to2,500 nm.

Instrument control and data processing arehandled effortlessly by our JASCO's user friendlyand innovative cross-platform software, SpectraManager™ II.

FVS-6000Vibrational Circular Dichroism

FP-8200 – FP-8300 – FP-8500 – FP-8600Spectrofluorometers

Designed with the latest technology, the FP-8000Series spectrofluorometers incorporate the highestsensitivity, fastest spectral scanning capability andexcellent analysis-oriented functionality offeringintegrated solutions for advanced materialsresearch and biochemical analysis applications.

To meet the most stringent analysis demands, avariety of accessories are available for integrationwith a range of sophisticated control and analysisapplications available in the user-friendly SpectraManager™ II software to offer a flexible platformfor any fluorescence and phosphorescenceapplication.

The FVS-6000 not only allows you to easilyobtain fingerprint VCD spectra, but also hasseveral unique features such as a measurementrange extension option of 4000-750 cm-1.

Since the CD signals in the infrared region areone or more orders of magnitude lower thanECD signals in the UV-Vis region, high sensitivityand stability are required for a VCDspectrometer.

The FVS-6000 is the VCD spectrometer of choicefor highly sensitive VCD measurements.

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VIR-100 – VIR-200 – VIR-300Portable FT-IR Spectrometers

The VIR-100/200/300 series are compact,lightweight, flexible FT-IR systems.

The collimated entrance and exit ports make it anideal instrument for a wide range of applications.

The standard instrument includes a hermeticallysealed interferometer, DLATGS detector, highintensity source, KRS-5 windows and automaticalignment. Options can be added for increasedsensitivity, optional spectral ranges including NIR,and battery operation.

For even greater flexibility, external connectionoptics allows the user to install up to three differentattachments in one system, selecting the mostappropriate application accessory by simplyswitching the PC controlled optical configuration.

RMP-510 – RMP-520 – RMP-530Portable RAMAN Spectrometers


JASCO’s new RMP-500 Series has beendeveloped to meet the needs of MaterialScience, Manufacturing and Biochemistry bycombining the flexibility of a fiber optic probewith a portable Raman Microspectrometer.

The RMP-500 Series consists of three models,RMP-510, RMP-520, RMP-530 ranging fromsmall, portable units suitable for in-situmeasurements to research-grade systems thatwill meet even the most difficult applicationrequirements.

The RMP-500 Series portable Ramanspectrometer systems feature an integrated fiberoptic probe with a small X-Y-Z stage, a compactlaser, a high-throughput spectrograph and CCDdetector.

JASCO is the first manufacturer to develop apowerful, cross-platform software package,“Spectra Manager”, for controlling a wide range ofspectroscopic instrumentation. Spectra Managerprogram is a comprehensive package for capturingand processing data, eliminating the need to learnmultiple software packages and offering the user ashallower learning curve.

Several types of measurement data files (UV-Vis/NIR, FT-IR, Fluorescence, etc.) can be viewed in a singlewindow, and processed using a full range of data manipulation functions.

The latest version, Spectra Manager II, includes four measurement programs, a spectra analysis program, aninstrument validation program and the JASCO Canvas program as standard. It is possible to analyze dataeven during sample measurements.

Spectra Manager CFR provides features to support laboratories in compliance with 21 CFR Part 11.

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JASCO has the largest range of optical detectors -from dual wavelenght UV to diode array to uniquechiral detectors. All the detector are designed tomeet U-HPLC requirements, data acquisition rate of100Hz.

SFC-4000Supercritical Fluid Chromatography

The JASCO SFC/SFE 4000 integrated Analytical SFCsystem has been developed for all aspects ofanalytical SFC; including routine separation, methoddevelopment and small scale preparation ofsamples at the mg scale.

With a simple intuitive software and robustengineering, the JASCO SFC system is a powerfultool for analytical separations.

LC-4000High Performance Liquid Chromatography

Chromatography Product Portfolio

The LC-4000 Series is the latest in a long historyof innovative HPLC systems developed by JASCOreaching all the way back to the start ofcommercial HPLC in the early 1970s.

The concept of the integrated LC-4000 seriesHPLC provides key separation platforms at50MPa, 70MPa and 130MPa which correspondto conventional HPLC, the increasingly popularRapid Analysis Fast HPLC and sub 2um U-HPLCrespectively.

Each platform is supplied with a dedicated pumpand autosampler matched to the operatingpressure and share detectors optimized for high-speed 100Hz acquisition and the narrow peakshapes common to both Fast HPLC and U-HPLC.

In the LC-4000 series, SSQD technology (SlowSuction, Quick Delivery) has been re-developed,with a completely new solvent deliverymechanism offering the highest stability insolvent delivery across the entire analytical flowrate range used in the PU-4100 Fast HPLC andPU-4200 U-HPLC pump models.

Both HPLC and SFC/SFE systems are coupled withChromNAV 2.0 data system to offer both HPLC andspectral data handling for most of the detectorseven with the dual wavelength UV detector.

A newly added feature of ChromNAV 2.0 is theautomatic e-mail notification on yoursmartphone/tablet, stay always updated on analysisstatus of your LC-4000. Full GLP compliance and 21CFR part 11.

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Table of Contents

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Application Description Page Solutions

UV-0014-EChromaticity and Turbidity Quantitative Measurement using UV/Vis Spectrophotometer

11 Enviromental

UV-0015-EQuantitative Measurement of Chromium According to RoHS Directiveand Hexahydric Chromium Treated to Screws

13 Enviromental

JE-Ap-UV1005-0007 Absolute Water Analysis with 30 cm Cell 15 Enviromental

JE-Ap-UV1005-0008 Quantitative Determination of Chromium by Diphenylcarbazide Method 17 Enviromental

UV-0001-E TCH-703 8-position Turret Micro Cell Holder 20 Pharmaceutical

UV-0003-E Quantitative Determination of Proteins 22 Pharmaceutical

UV-0008-E DNA melting by One Drop measurement using capillary jacket 28 Pharmaceutical

UV-0016-EDNA Melting Measurement with the PAC-743/743R Water-cooled Peltier Cell Changer Measurement with a Temperature Sensor in Cell

30 Pharmaceutical

UV-0017-EDNA Melting Measurements (3) with the PAC-743/ 743R Water-cooled Peltier Cell Changer “1 mm 10 μL 8-Position Micro Cell Block”

32 Pharmaceutical

UV-0018-EThe Model SAH-769 One-Drop: A Dedicated Accessory for Extremely Small Amounts of Protein and Nucleic Acid Samples

34 Pharmaceutical

JE-Ap-UV1005-0006 Melting Experiments with V-600 Spectrophotometers Series 37 Pharmaceutical

UV-0019Measurement of the reduction reaction of 2,6-dichloroindophenol (DCIP)using the absorption stopped-flow system

39 Pharmaceutical

UV-0024 Transmission measurement of Volvox by using MSV-5000 series 40 Pharmaceutical

UV-01-04 Automated Sun Protection Factor (SPF) Analysis of Sunscreen using UV-Vis 42 Petro & Chemicals

UV-0011-ECountermeasure technique against heat island phenomenon - Evaluation of solar reflective paint material -

43 Petro & Chemicals

UV-0036 Evaluation of sun protection fabrics by using a UPF evaluation system 45 Petro & Chemicals

UV-0033Evaluation of the privacy film using an automated absolute reflectance measurement accessory


UV-0023 Thickness Analysis of natural oxide film on microscopic Si pattern 49 Petro & Chemicals

JE-Ap-UV1005-0006 Luminous Color Measurement by using UV/Vis Spectrophotometer 50 Petro & Chemicals

JE-UV-02-16 Bandgap Measurements for Titanium Dioxide Powder 54 Petro & Chemicals

JE-UV-03-16 Reflection Object Color Measurements using Color Diagnosis System 55 Petro & Chemicals

JE-UV-04-16 Gardner Color Measurement System 57 Petro & Chemicals

JE-UV-05-16 Hazen Color Measurement System 58 Petro & Chemicals

JE-UV-06-16 Color Analysis System for RGB Color Filters 59 Petro & Chemicals

UV-0025Estimation of refractive index of monocrystalline sapphire by polarization measurement using MSV-5000 series


JE-UV-01-16 Transmittance and Reflectance Measurement System for Minute Lenses 62 Petro & Chemicals

UV-0040Evaluation of antireflection film by using absolute reflectance measurement accessory


UV-0042Introduction of Haze value measurement system for UV/Vis spectrophotometer


UV-0043Example of the measurement of a highly reflective material, a dielectricmulti-layer mirror using absolute reflectance spectroscopy


UV-0044 Measurement reproducibility of Color Diagnosis System 70 Petro & Chemicals

Application UV-0014-E

Chromaticity and Turbidity Quantitative Measurement using UV/Vis Spectrophotometer

In this Application Note, the method formeasurement of chromaticity and turbidity usingUV/Vis spectrophotometer based on clean water testmethod will be described.

ChromaticityChromaticity measurement is to check the colorationdegree of clean water and wastewater using humicacid.This test is applied by observing the absorption at390 nm which shows the yellow color of humic acidby using UV/Vis spectrophotometer.

Measurement/analysis system• V-630/650/660/670 UV/Vis spectrophotometer• LSE-701 Long path cell holder• VWWQ-789 Chromaticity/turbidity measurement

program• Rectangular cell 50 mm or 100 mm

Standard sampleCobalt chloroplatinate which has a color similar toyellow-brown of humin is used as standard samplefor chromaticity. 2.49 g of Potassium chloroplatinateand 2.02 g of cobalt chloride are dissolved in 200 mLof hydrochloric acid, and then purified water isadded to make the solution of total volume 1 L. Thissolution is neat standard sample with chromaticity1000 degree.

Test methodMeasuring the absorbance of sample in cell of 50mm or 100 mm pathlength at the wavelength of 390nm.

Procedure1. Standard solution is prepared from neat standard

sample diluted by purified water. Blank sample ispurified water filtrated using 0.2 μm membranefilter.

2. Chromaticity calibration curve is created from themeasurement results of blank sample and standardsolution prepared in 1.

3. Sample water is filtrated using membrane filter orcentrifuged and the supernatant is used as sample.

4. The absorption of sample prepared in 3 at 390 nm ismeasured, and chromaticity is calculated from theresults and calibration curve.

Figure 1 Chromaticity calibration curve

Concentration[ Chromaticity ]

Absorbance Quantitative Value[ Chromaticity ]

0 0.000 -0.06

0.5 0.004 0.51

1 0.007 0.97

2 0.015 2.04

20 0.141 20.07

50 0.350 49.99

100 0.699 99.99

Table 1 Chromaticity calibration curve

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In Standard solutions with chromaticity at 0, 0.5, 1, 2,20, 50, 100 degree were measured using 50 mm lightpathlength cell and these results are shown in thecenter column of Table 1. Measured absorbancevalues are input to the calibration curve and thecalculated quantitative values of chromaticity areshown in the right column of Table 1. From theabove results, the standard deviation (σ) betweenthe obtained quantitative value and actualchromaticity is 0.04(6) degree, detection limit, 0.15and quantitation limit, 0.46 degree.*1)

*1) Detection limit is calculated from 3.3σ andquantitative limit is calculated from 10σ.

Calibration curve information: y = 0.0070x +0.0004(5) R2 = 1.0000

TurbidityIn turbidity measurement, the turbidity degree dueto insoluble particles, microbe and organic substancein clean water and wastewater is tested. Scatteringlight at 660 nm is measured using UV/Visspectrophotometer in transmission measurementmethod or integrating sphere photoelectricspectrophotometry.

Standard sampleImmixture polystyrene suspension is used asstandard sample of turbidity. Mixture of 5 kinds ofpolystyrene particles shown in the Table 2 is statedas neat solution of turbidity at 100 degree, which iscommercially available.

Measurement/analysis system• V-630/650/660/670 UV/Vis spectrophotometer• LSE-701 Long path cell holder• Quantitative measurement program• Rectangular cell 20 mm, 50mm and 100 mm

Procedure1. Neat sample solution is diluted by purified water and

prepared as standard solution. Purified water filtrated using 0.2 um membrane filter is used as blank sample.

2. Turbidity calibration curve is created from the measurement results of blank sample and standard solution prepared in 1.

3. The absorbance of sample water at 660 nm is measured, and turbidity is calculated from the results and calibration curve.

Figure 2 Turbidity calibration curve(Transmission measurement method)

Category Normal diameter (um) Mixture Ration (%)

No. 6 0.5 6

No. 7 1 17

No. 8 2 36

No. 9 5 29

No. 10 10 12

Table 2 Polystyrene standard particle of turbidity at 100

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Standard solutions with turbidity at 0, 5, 10, 50, 100 degree were measured using 20 mm light pathlength cell and the results are shown in Table 3. By the same method as chromaticity, the standard deviation is calculated as 0.36 degree, detection limit as 1.18 and quantitation limit as 3.6 degree.

Calibration curve information: y = 0.0050x + 0.0014 R2 = 1.0000

Standard solutions with turbidity at 0, 0.5, 1, 2, 5, 10degree were measured using 20 mm light pathlength celland the results are shown in Table 4. From thecalibration curve, the standard deviation is calculated as0.08(6) degree, detection limit as 0.28 and quantitationlimit as 0.86 degree.*2)

*2) 50 mm light pathlength rectangular cell is recommendto be used for the analysis of low turbidity sample withconcentration close to or less than quantitation limit .

Calibration curve information: y = 0.7018x + 0.0397 R2 =0.9997

Concentration[ Turbidity ]

Absorbance Quantitative Value[ Turbidity ]

0 0.000 -0.27

5 0.026 4.81

10 0.052 10.07

20 0.102 20.09

50 0.256 50.64

100 0.502 99.67

Table 3 Turbidity calibration curve (transmission measurement method)

Integrating sphere photoelectric spectroscopyMeasurement/analysis system• V-650/660/670 UV/Vis spectrophotometer• ISV-722/ISN-723 Integrating sphere unit• VWWQ-789 Chromaticity/turbidity measurement

program• Rectangular cell 10 mm, 20 mm, 30 mm and 50

mm

Procedure1. Standard solution is prepared from neat standard

sample diluted by purified water. Blank sample ispurified water filtrated using 0.2 um membranefilter.

2. Turbidity calibration curve is created from themeasurement results of blank sample andstandard solution prepared in Firstly, standardwhite plate is mounted in integrating sphere andtotal light transmittance (Tt) is measured, andthen the plate is removed and diffusetransmittance (Td) is measured.

3. Tt and Td of sample water at 660 nm ismeasured, and turbidity is calculated from theresults and calibration curve.

Figure 3 Measurement using integrating sphere

Figure 4 Turbidity calibration curve(Integrating sphere photoelectric spectroscopy)

Concentration[ Turbidity ]

Td/Tt x 100 Quantitative Value[ Turbidity ]

0 0.004 -0.05

0.5 0.389 0.50

1 0.726 0.98

2 1.435 1.99

5 3.666 5.17

10 7.003 9.92

Table 3 Turbidity calibration curve (transmission measurement method)

1). Measurement of total light transmittance (Tt)

(2). Measurement of diffuse transmittance (Td)

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Quantitative Measurement of Chromium According to RoHS Directiveand Hexahydric Chromium Treated to Screws

Hexahydric chromium (Cr(VI)) is one of the regulatedmaterials by RoHS directive. In this application note,the measurement example of two kinds of screwswith chromate treatment is introduced.

1. SampleSteel screws A: pan sems, spring washer + bigwasher, M3x6, 8 pieces, 5.7 gSteel screws B: pan-head, M4x12, 19 pieces, 14.0 g

2. Measurement ProceduresChromium was extracted by hot water and measuredits quantity by chromogenic reaction ofdiphenylcarbazide method.

3. Chromium ExtractionWell-known extraction methods of chromium are theHydrothermal Extraction (JIS H 8625), the AlkalineDecomposition (EPA 3060A), the Shaking Extraction(DIN53314), and the Volvo method.The Hydrothermal Extraction of JIS H 8625 is awidely-used easy method for extracting chromiumtreated to electrical and manufacturing products.The method was applied to the steel screws toextract chromium.

Extraction solvent: Purified Water 25 mLExtraction temperature: 80 degrees *1)

Extraction time: 30 minutes *1)

*1) In the JIS H 8625, the extraction temperature is 100degrees and the time is 5 minutes. The extraction herewas performed by using thermostat bath at 80 degrees.The time for extraction was 30 minutes.Although this condition was determined by confirmingthe end of the Cr(VI) chromium extraction, the conditionmay change for the same kind of screws because thechromium coatings differ for each screw.Confirming the shape of spectrum, there were nocontaminations of interference substances with theextraction condition.

4. Chromogenic Reaction of Cr(VI)After hydrothermal extraction, screws were picked out from the sample vial. The extraction liquid was cooled to room temperature. Then, the chromogenic reagent of KYORITSU CHEMICAL-CHECK Lab (Figure 3) was added to the extraction liquid. The liquid was stirred for 1 minute and left to stand for 5 minutes.

Figure 1 Samples

Figure 2 Measurement procedures

Figure 3 Reagent set for water analyzer No.31- Cr (VI)

(KYORITSU CHEMICAL-CHECK Lab)

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Quantitative Measurement of Chromium According to RoHS Directiveand Hexahydric Chromium Treated to Screws

5. Quantitative MeasurementAfter leaving the sample for 5 minutes, turbidity wasobserved (Figure 4).The untouched sample and filtered sample weremeasured to compare their spectra.

Instrument• JASCO V-630 spectrophotometer• 10 mm rectangular quartz cell

Measurement ParametersMode: AbsMeasurement Range: 650 - 400 nmData Interval: 0.2 nmUV/Vis bandwidth: 1.5 nmResponse: MediumScan Speed: 400 nm/min

6. Measurement ResultsThe spectra of untouched and filtered samples areillustrated in Figure 5. The comparison of two spectrashows higher baseline for the spectrum of untouchedsample. This is caused by the dispersion by theinterference substances. Three wavelengths quantitativeanalysis (peak wavelength: 542 nm, base wavelength:635 and 402 nm) was considered to confirm the effectsof the interference substances. Table 1 shows the resultsof the peak height calculations. Regardless of thepretreatment, almost same results can be obtained.

7. Quantitative ResultsThree wavelengths quantitative analysis was examinedon the peak height calculated with peak wavelength at402 nm and base wavelengths at 635 and 542 nm. Thecalibration curve obtained by the diphenylcarbazidemethod in UV application data Vol.2 was applied to thequantitative analysis. Table 2 shows concentration ofCr(VI) for the sample A and sample B.The quantitative results in Table 2 show the two-sided95% confidence interval. Both sample A and B have theconfidence interval +/-0.005 mg/L against thequantitative values.With the method introduced here with JASCOspectrophotometer provides high precisionmeasurement.

Figure 4 Sample after chromogenic reactionUntouched sample (left) Filtered sample (right)

Sample A Sample B

Untouched Filtered Untouched Filtered

One WL Absorbance at 542 nm 0.10393 0.09288 0.06365 0.04482

Three WLPeak height at 542 nm

Base wavelenght: 402, 635 nm0.08782 0.08765 0.03932 0.03915

Table 1 Results of peak height calculation

Sample A Sample B

Untouched Filtered Untouched Filtered

Quantitative Results 0.135 ±0.006 0.135 ±0.006 0.059 ±0.005 0.059 ±0.005

Table 2 Quantitative Results of Cr(VI) (mg/L)Figure 5 Spectras

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Application JE-Ap-UV1005-0007

Absolute Water Analysis with 30 cm Cell

Superficially clean waters are analyzed in the field ofnot only inspection of drinking and environmentalwater, but quality control of glass.Of them, test of drinking water has water coloranalysis since it tells the concentration of humicmaterials that is precursor substance oftrihalomethane.The simplest method of color analysis defined intesting method of drinking water is visual check withcolor comparison tubes, however, results of themethod depend on “observer’s aesthesia”.Here, JASCO introduces new method to analyze“Superficially clean water” with UV/Visspectrophotometer and light path length 30 cm cell.

1. System Configuration• V-650/660/670 spectrophotometer• Extended sample compartment• 30 cm cylindrical cell

2. Introduction of 30 cm CellJASCO extended sample compartment is dedicatedfor light path length 30 cm cylindrical cell.The 30 cm cell enables to measure samples with tinyabsorbance precisely that cannot be measured byusual cell.An integrating sphere is mounted in thecompartment to detect any size of light beamaccurately. Easy measurement is now offered to youby just putting samples in the 30 cm cell andmounting it in the sample compartment.

3. SamplesUltra pure water, purified water, tap water,groundwater, and river water.

4. Measurement ParametersMeasurement Range: 850 - 220 nmData Pitch: 0.5 nmUV/Vis Band Width: L5.0 nmResponse: MediumScan Speed: 400 nm/min

5. Measurement and AnalysisEach sample was measured with 30 cm cell having airbaseline. Ultra pure water indicated low absorbance atthe all wavelength range (Figure 2). Since we regardedultra pure water as almost clear, employed it to baselineto cancel the reflection or absorption of cell andobtained spectrum for each sample (Figure 3).

The results obtained with 30 cm cell was analyzed withthe [Color Diagnosis] program, and then plotted on thechromaticity diagram (Figure 4). Same samples weremeasured with 10 mm cell and same calculation wasproceeded (Figure 5).Comparison between two chromaticity diagrams, colorsthat cannot be defined with 10 mm cell is clear with 30cm cell. From the result, river water is yellower than theother waters and it is thought of as containing humicmaterials.Moreover, in the order of groundwater, tap water, andpurified water, they come close to the light source that iswhite light.

Figure 1 Spectrophotometer with extended sample compartment

15


Absolute Water Analysis with 30 cm Cell

With JASCO 30 cm cell, the color of “Superficially clearwater” can be measured. It is available for themeasurement of low concentration samples and in widevariety of water analysis fields. Extended samplecompartment can be applied not only for liquid samples,but clear baculiform solid samples such as fibers.

From environmental analysis to quality control, JASCO extended sample compartment with 30 cm cell is useful in wide variety of fields.

Figure 2 Absorbance spectrum of ultra pure water (red)

Figure 3 Absorbance spectra of samples

Figure 4 Chromaticity diagram of results measured by 30 cm cell

Figure 5 Chromaticity diagram of results measured by 10 mm cell

Measurement Transmittance

Wavelength Range250 to 800nm (UV/VIS)250 to 2000 nm (NIR)

Detector 60mm diam. Integrating Sphere

Cell Size30mm diam.Light path length: 30cmWindow: Quartz

Specification of Extended Sample Compartment with 30 cm Cell

16


Quantitative Determination of Chromium by Diphenylcarbazide Method

Chromium is widely used for metal materials such asiron and steel, chromium alloy, and refractoryproducts. Plating, tanning, and pigments use thechromium as well. Chromium is very useful for ourindustrial society, however, against human body,chromium ions impinge as cell poison. For thatreason, chromium requires careful handling andquantitative determination. The most widely usedquantitative method is the diphenycarbazideabsorptiometrical method with spectrophotometer.The detection and determination limits of chromiumwere reviewed here with JASCO V-630spectrophotometer. Calibration curves were createdby using chromogenic kit of reagent manufacturersfor easy quantitative determination.

1. Reagents• Reagent set for water analyzer No.31- Cr (VI)

(KYORITSU CHEMICAL-CHECK Lab)• Special grade chemicals K2Cr2O7• Distilled water

2. Instrument• JASCO V-630 spectrophotometer• 10-mm rectangular cell: Quartz

3. Reagents preparation3.1 Standard SolutionK2Cr2O7 of special grade chemicals was dried at 150°Cfor one hour and left in a desiccator, then 8.79 mg of theK2Cr2O7 were weighed out. In a 100-mL measuring flask,the K2Cr2O7 was dissolved in water and diluted to 100mL. 80.4 mL of the solution was weighed out to theother 100-mL measuring flask and diluted to 100 mL.The diluted solution was a standard stock solution of25.0 mg/L Cr (VI). The stock solution was diluted 100times to prepare a standard solution of 0.25 mg/L.

3.2 Solutions for calibration curveIn accordance with Table 1, distilled water and standardstock solution were weighed out by pipettes and mixedto prepare 25 mL solutions.

Concentration(mg/L)

Stock Solution(µL)

DW(mL)

Concentration(mg/L)

Stock Solution(µL)

DW(mL)

1.500 1500 23.5 0.00750 750 24.3

1.000 1000 24.0 0.00500 500 24.5

0.500 500 24.5 0.00250 250 24.8

0.250 250 24.8 0.00125 125 24.9

0.125 125 24.9 0.00100 100 25.0

0.060 60 25.0 0.00000 0 25.0

0.040 40 25.0 - -

0.020 20 25.0 - -

0.010 10 25.0 - -

Table 1 Solutions for calibration curves*1)

*1) Solutions from 0.010 to 1.5 mg/L were prepared with the standard stock solution of 25.0 mg/L [Cr (VI)]. Solutions from 0.0010 to 0.0075 mg/L were prepared with standard solution of 0.25 mg/L.

17



4. Chromogenic ReactionChromogenic reaction was processed at the roomtemperature (21°C). One package of the reagent setfor water analyzer No.31- Cr (VI) was added to thecalibration curve solutions right after opening it*2).The bottle of solution was agitated by hand forapproximately 60 seconds*3). Dissolution of thereagent was confirmed *4).

4.1 Reaction TimeCr (VI) solution of 25 mL with the concentration of0.5 mg/L was prepared in order to determine thetime from addition of the reagent to themeasurement. The time course of the chromogenicreaction was observed. The reagent set for wateranalyzer No.31- Cr (VI) was added to the solutionright after opening it and agitated for one minute,and then the time course of the absorption wasmeasured at the wavelength of 540 nm. Here, 90seconds were assumed as the time spent untilmeasurement after addition of the reagent. Thechromogenic reaction was completed 150 secondsafter starting measurement (4 mins after addition ofthe reagent). The color became stable 210 secondsafter starting measurement (5 mins after addition ofthe reagent).

4.2 MeasurementThe Spectra Measurement Program was used for thisexperiment. The reagent was added to the solution andagitated for one minute, and then it was measured afterleaving for 5 minutes.The light path of the reference side was empty.Baseline was measured with water in the cell used forsample measurement.

Photometric Mode AbsMeasurement range 650 - 400 nmData pitch 0.2 nmBand width (UV/Vis) 1.5 nmResponse MediumScanning speed 400 nm/min

Figure 1 Time course measurement at 540 nm

Figure 2 Color Changing

Figure 3 Absorption spectra of Cr (VI) solutions

with concentration from 0.01 mg/L to 1.5 mg/L

*2) The solubility of the reagent set deteriorates with time after opening it, since the reagent absorbs moisture.*3) Agitate the solution well, since the inadequate agitation impedes the smooth chromogenic reaction and has effect on the absorbance.*4) Granular reagent remained even if the reagent dissolved perfectly in the solution. The remainder, however, was precipitated at the bottom

of cell and did not have influence on the measurement.18



5. ResultsThe fluctuation of the bases occurred for Cr (VI)solution with the concentration between 0.01 mg/Land 0.001 mg/L. For that reason, the calibrationcurves were created with the peak height at 542 nmwhen the bases of wavelength were set at 402 nmand 635 nm. The determination limit of thecalibration curve that was created with allconcentration range was 0.09 mg/L.This shows that JASCO V-630 can determine thequantity of the chromium with the concentrationrange between 0.09 mg/L to 1.5 mg/L. Furthermore,when the calibration curve was created with the dataof the Cr (VI) solution with the low concentrationrange between 0.01 mg/L and 0.001 mg/L, thedetermination limit was 0.0025 mg/L that proveshigher sensitivity of the JASCO V-630.

Y=A * X + BA=0.63663±0.00621B=0.00183±0.00303Correlation Coefficient =0.99987Standard Error=0.00749Detection limit=0.00477 mg/LDetermination limit=0.08835 mg/L

Y=A * X + BA= 0.62824±0.02099B= -0.00018±0.00011Correlation Coefficient= 0.99958Standard Error= 0.00012Detection limit = 0.00017 mg/LDetermination limit= 0.00250 mg/L

Concentration(mg/L)

Abs Concentration(mg/L)

Abs

0 -0.00019 0.02 0.013

0.001 0.00036 0.04 0.02695

0.00125 0.00068 0.06 0.03932

0.0025 0.00134 0.125 0.0827

0.005 0.00302 0.25 0.17008

0.0075 0.00459 0.5 0.33018

0.01 0.00603 1 0.64278

- - 1.5 0.94904

Table 2 Concentration and Absorption

19


TCH-703 8-position Turret Micro Cell Holder

The TCH-703 eight-position turret micro-cellaccessory is an attachment designed specifically forthe V-630/630BIO instruments to provide aconsistent measurement of eight samples with aminimum sample quantity of 4 μL.It is a simple procedure to measure 8 samples as amaximum: pipette samples into the removablesample turret (Figure 1), set the turret cell into theholder (Figure 2), turn the knob to place a sampleinto the sample beam (Figure 3), and then press thestart button. This attachment is very useful for themeasurement of limited sample quantities for DNAand/or protein concentrations.

Measurement accuracy

Measurement accuracy of the 8 cellsTo verify the measurement accuracy of the turret cells,analysis of DNA samples of raw bovine thymus wasperformed in the 8 cells at specific concentrations,measuring the quantity using the Warburg-Christianmethod. The CV’s for these measurements were 2%when the DNA concentration was 50 μg/mL, 11% for 10μg/mL, and 10% for 5 μg/mL.

Reproducibility of the turret rotationThe same samples were measured while the turret wasrotated to analyze the variation of the quantitativemeasurement in the same cell. The CV for thesemeasurements was 0.3% when the DNA concentrationwas 50 μg/mL, 1.7% for a 10 μg/mL concentration, and3.5% for 5 μg/mL (Table 1).

Figure 1

Figure 2

Figure 3

DNA 50μg/mL

DNA 10μg/mL

DNA 5μg/mL20


TCH-703 8-position Turret Micro Cell Holder

Detection limit and determination limitThe quantitative results outlined in Table 1 wereused to create a calibration curve of the quantitativeresults vs. concentration (Figure 4). This calibrationcurve has a calculated detection limit of 0.38 μg/mLand a determination limit of 6.4 μg/mL. Thedefinition of the detection limit used here is “theconcentration calculated from the calibration curvewhen the maximum quantitative value of the 95%confidence interval is read at a calculatedconcentration of 0 μg/mL”. The definition of thedetermination limit is “the concentration calculatedfrom the calibration curve using some specificquantitative results that has a coefficient of variationof 10%”.

Sample InfusionA round-shaped gel loading tip is the most appropriatepipette tip to load samples into the turret cells. Using thegel loading tip prevents air bubbles in the cells.

Cleaning the turret cellA round-shaped gel loading tip is used for both sampleinfusion and cleaning the cell with a cleaning solventsuch as water. To remove protein samples, commercialdetergents for instrument cuvettes can be used. If theturret cells are heavily contaminated, a concentratednitric acid solution can also be used. JASCO offers awashing device, the μWash, which is designed to cleanthe cell easily using water, methanol, ethanol, and othersolvents.

Washing accessory for micro cells such as 8-positionTurret Micro Cell Holder

• Insert the needle tip into the cell. Pushing the buttonson the syringe several times cleans the cell using a ‘filland drain’ action.

• The Wash device can wash the cell with a smallquantity of a washing solution.

• The MW-2000 is applicable to the sample volume lessthan 100 μL.

Figure 4 Calibration curve of the quantitative results vs. concentration

Y = A x E + BA = 0.824697 +/- 0.010735B = 0.236037 +/- 0.311315Correlation coefficient = 0.998451Standard error = 1.12974Detection limit = 0.37749Determination limit = 6.44095

Specifications

Needle size 0.7 mm O.D. x Approx. 10 mm (taper type)

Maximum injectionand suction

Approx. 100 μL

Bottle volume 10 mL

Materials PTFE, PP, PE, EPDM, Silicone, Stainless

Cleaning solventWater, Methanol, Ethanol and other volatile solvents

21


Quantitative Determination of Proteins

Generally, the concentration of proteins is measuredusing UV-Vis spectrophotometers. The JASCO V-630Bio is a UV/Vis spectrophotometer designed forbiochemical analysis.The V-630 Bio is equipped with 6 quantitativecalibration curves based on UV absorptionspectrophotometry including the Lowry, Biuret, BCA,Bradford, and WST methods.

Table 1 shows the features of the six differentquantitation methods. As outlined, the preferredmethod can be selected by reviewing the sample andrange of the quantitation and existence of anypossible contaminants.Five of the analysis methods are quantitativemethods that utilize a chromogenic reaction.Reagent manufacturers produce chromogenic kits forBCA, Bradford or WST with an instruction manualexplaining the measurement procedures*1). On theother hand, the chromogenic reagents for the Lowryand Biuret methods need to be prepared by the user.The measurement procedures for the Lowry andBiuret methods differ depending on each document.For that reason, this application data explains how tocreate the calibration curves for the Lowry, Biuretand UV absorption analysis methods. Fluctuations ofthe values depending on the type of proteins and thedifferences of the concentration ranges calculated asa result of the cell volume*2) were also investigated.

Figure 2 Quantitative Analysis program—

Set Calibration Curve dialog

Figure 3 Disposable cuvette

22



Principle Concentration range Advantages Disadvantages

UV Abs

The absorption maximum at 280nm corresponds to the responseof the tyrosine and tryptophanand is used for the analysismethod.

50 to 2000 μg/mL (*BSA)Simple method.Sample can be useafter measurement.

The absorbance differs for eachprotein. Proteins such ascollagen and gelatin that do nothave absorption at 280 nmcannot be measured.Contamination by nucleic acidswith absorption in the UV regionobscures the measurement.

Biuret

Protein solutions turn purple afterthe polypeptide chain chelateswith a copper ion. An alkalinesolution of Biuret reagentincluding copper sulfate andRochelle salt is added to a proteinsolution. Uses the absorptionmaximum at 540 nm todetermine the quantity.

150 to 9000 μg/mL (BSA)

Simple procedure.The chromogenicrate is constant formost proteins.

Low sensitivity. Sample with alow protein concentrationcannot be measured.The chromogenic reaction isinfluenced by highconcentrations oftrisaminomethane, amino acids,and ammonium ion.

Lowry

Add an alkaline copper solution toa protein solution. Tyrosine,tryptophan, and cisteine ofproteins reduce molybdenumacid and phosphotungstic acid ofa phenol reagent, turning thesolution blue. Uses the absorptionmaximum at 750 nm todetermine the quantity.

5 to 200 μg/mL (BSA)High sensitivity.Widely used.

Complicated procedure with along preparation. Since thechromogenic reaction occurs bya reduction reaction,contamination of the reductionmaterial interferes with thequantitative determination. Thechromogenic rate differs foreach protein.

BCA

The BCA method combines theBiuret method and BicinchoninicAcid (BCA). BCA has highsensitivity and selectivity forcopper ions. When a copper ionthat is formed by the reductionaction of protein reacts with 2molecules of the BCA, thesolution turns purple. Uses theabsorption maximum at 560 nmto determine the quantity.

20 to 2000 μg/mL (BSA)

Simple procedure.High sensitivity withwide Concentrationrange.

Thiol, phospholipid, andammonium sulfate interfereswith the measurement.

Bradford

The absorption maximum of aprotein shifts from 465 nm to 600nm when the protein binds to theCoomassie Brilliant Blue G250,one of triphenylmethanse bluepigment. Uses the absorptionmaximum at 600 nm todetermine the quantity.

10 to 2000 μg/mL (BSA)

Very simpleoperation. Hardlyinfluenced by theblocking materials

The chromogenic rate differs foreach protein. Contamination bya surfactant can interfere withthe chromogenic reaction.

WST

Reduce WST-8 with proteins witha high pH, turning the sampleblue. Uses the absorptionmaximum at 650 nm todetermine the quantity.

50 to 5000 μg/mL (BSA)Simple operation.Hardly influenced bya surfactant.

The chromogenic rate differs foreach protein.

23



1. UV Absorption MethodFigure 4 illustrates the absorption spectrum ofHuman Serum Albumin (HSA). Simple UV Absorptionspectrophotometry can determine the quantity ofproteins in the sample by using the maximumabsorption at 280 nm.

1.1 SamplesBovine Serum Albumin (BSA): 0.02, 0.025, 0.05, 0.1,0.2, 0.25, 0.4, 0.5, 1, (1.5, 2) mg/mLHen Egg Lysozyme: 0.02, 0.025, 0.05, 0.1, 0.2, 0.25,(0.4, 0.5) mg/mLChymotrypsin from bovine pancreas: 0, 0.1, 0.2,0.25, 0.4, 0.5 mg/mL

Bracketed values are for concentrations used with the 10mm rectangular cell. The other values are concentrationsused with the 10 mm rectangular cell and micro cell.

1.2 Measurement ProceduresMeasure the absorbance of the protein solution usingthe wavelength of 280 nm.

Figure 4 Absorption spectrum of HSA

Table 2 Results for the UV absorption method

24



2. Biuret MethodProtein solutions turn purple with the absorptionmaximum of 540 nm when Biuret reagent is added(Figure 5).The time course of the chromogenic reaction wasmeasured at 540 nm absorption. The absorbancebecame stable approximately after 60 minutes(Figure 6). Figure 7 demonstrates the spectra of theHSA aqueous solutions that were measured after 60minutes.Biuret method determines the quantity by using theabsorption maximum at 540 nm after reacting withthe sample for 60 minutes.

2.1 Chromogenic ReagentBiuret reagent: Add 60 mL of 10% NaOH to 100 mL ofaqueous solution to dissolve 0.3 g CuSO4 and 1.2 gRochelle salt, and then add water to 200 mL. (Standardreagent can be saved in a polyethylene bottle.)

2.2 SamplesBovine Serum Albumin (BSA): 0, 0.25, 0.5, 1, 5, 9 mg/mL(10 mm rectangular cell (quartz))0, 1, 3, 5, 9 mg/mL (Micro cell)Hen Egg Lysozyme: 0, 1, 3, 5, 9 mg/mL (10 mmrectangular cell (quartz), Micro cell)Chymotrypsin from bovine pancreas: 0, 1, 3, 5, 9 mg/mL(10 mm rectangular cell (quartz), Micro cell)

2.3 Measurement ProceduresAdd Biuret reagent (2.0 mg) to 500 μL of proteinaqueous solution and mix them well. Then react thesolution for 60 minutes. Measure the absorbance at thewavelength of 540 nm.

Figure 5 Color change after adding

Biuret regent

Figure 6 Time course trace of the chromogenicreaction

Figure 7 Spectra of the HSA solutions using

the Biuret method

25



3. Lowry methodProtein solutions turn blue with an absorptionmaximum of 750 nm when Alkline copper solutionand Phenol reagent are added to the protein solution(Figure 8).The time course of the chromogenic reaction wasmeasured at 750 nm absorption. The absorbancebecame stable approximately after 60 minutes(Figure 9).Figure 10 illustrates the spectra of the HSA aqueoussolutions that were measured after 60 minutes.Lowry method determines the quantity by using theabsorption maximum of 750 nm after reaction of thesample for 60 minutes.

Table 3 Results for the Biuret method

Figure 8 Color change after adding Phenol reagent

Figure 9 The time course trace of the chromogenic reaction

26



*4) 0.5% CuSO4 solution: dissolve copper (II) sulfatepentahydrate (50 mg) and potassium sodium tartratetetrahydrate (0.1 g) in 10mL of water.

Phenol reagent: Dilute commercial solution (2N phenolchemical reagent of Kanto Chemical) to 1N.

3.2 SamplesSame concentrations are used both for the 10 mm rectangular cell and the micro cell of 10 mmBovine Serum Albumin (BSA): 0, 2, 20, 50, 100, 200 µg/mLHen Egg Lysozyme: 0, 1, 5, 10, 20, 50, 100, 200 µg/mLα-Chymotrypsin from bovine pancreas: 0, 2, 20, 50, 100, 200 µg/mL

3.3 Measurement ProceduresAdd 2.5 mg of alkaline copper solution to 500 μL of

protein solution. Allow to react for 10 minutes aftermixing well. Then, add phenol reagent. Rapidly mix and allow the solution to react for 60 minutes. Measure the absorbance at 750 nm.

Figure 10 Spectra of HSA aqueous solution by Lowry method

3.1 ReagentsAlkaline copper solution: Mix 50 mL of 2% Na2CO3 solution*3) with 1 mL of 0.5% CuSO4 solution*4) (can only be used for immediate analysis).

*3) 2% Na2CO3 solution: dissolve anhydrous sodiumcarbonate (2 g) and caustic soda (0.4 g) in 100 mLwater.

Table 4 Results for the Lowry method

27


DNA melting by One Drop measurement using capillary jacket

Melting measurement is one of remarkablemeasurement methods in Biotechnology field such asDNA melting and thermal denaturation of Proteins.For those measurements, 10mm rectangular typecell has been generally used, which requires largevolume of sample for each measurement.By using JASCO’s water-cooled Peltier thermostattedcell holder PAC-743/743R with micro 8-position cell itis possible to measure sample with volume as smallas 10 μL *, however, for measurement of samplewith much lower volume, it has been wished for along time to have an accessory enabling suchrequirement.*Refer to DNA melting measurement #3 in using ofPAC-743/743R No.UV-0017-E.

As one of solutions, JASCO introduces an application“DNA melting by One Drop measurement method”using JASCO V-630 BIO Spectrophotometer withcapillary jacket for melting measurement, capillaryand adaptor.

Capillary jacket for melting measurementCapillary is disposal type quartz glass cell, whoseoptical pathlength is 0.5 mm and minimum samplevolume is 3 μL. After sampling by a capillaryphenomenon, both edges of capillary are lapped byseal, which helps to avoid volatilization of sample.Such capillary is inserted into capillary jacket formelting measurement to be mounted to 6 channelcell block of Peltier cell holder or to PAC-743/ PAC743R Water-cooled Peltier thermostatted cell holder.This capillary jacket has temperature sensor insertionport and temperature measurement in this porthelps to measure accurate actual temperature ofsample.

Measurement System• V-630 BIO UV/VIS Spectrophotometer• ETCS-761 Water-cooled Peltier thermostatted cell

holder with stirrer• OPS-515 Temperature sensor assy• MCB-100 Mini water circulation bath• Capillary jacket for melting measurement, adaptor,

capillary, seal material

Measurement ProgramTemperature ramping / DNA melting program ( Standardsoftware of V-630 Bio)

SamplePoly (dA-dT)-Poly(dA-dT) pH7 phosphoric acid buffer

Measurement ConditionStart setting: 3 seconds in the set temperature +/- 0.10degrees CData acquisition interval: 0.1 degrees CTemperature gradient: degrees C / minResponse: FastMeasurement wavelength: 260 nm

Figure1 Capillary

Figure 2 Capillary jacket (L) and Adaptor (R) for melting measurement.

28


DNA melting by One Drop measurement using capillary jacket

ResultFig. 3 shows the result of measurement of Poly (dA-dT)-Poly(dA-dT). The green spectrum is the measurementresult data by using 10 mm rectangle type cell and blueone is the result data by using capillary cell. Temperatureis measured by the temperature sensor inside ofcapillary jacket.As the result of calculation based on above data formelting temperature, the similar results have beenobtained for both measurement, such as 63.8 degrees Cin 10 mm rectangular type cell and 63.9 degrees C incapillary cell respectively. It obviously proves that themeasurement using small volume capillary is as reliableas measurement by general method using 10 mm cell.

Sampling by using capillary

Sealing both edges

Setting capillary jacket into cell holder

Setting temperature sensorand capillary into jacket

Cap

illar

yP

art

Figure 3 Melting data29


DNA Melting Measurement with the PAC-743/743R Water-cooled Peltier Cell Changer Measurement with a Temperature Sensor in Cell

For the melting measurement of DNA samples, atemperature sensor can be inserted into the samplecells and the actual temperatures of samples plottedon the horizontal axis in order to increase theaccuracy of the temperature readings for the meltingexperiment. This measurement technique is easy tobe applied for 10-mm rectangular cells with a largersample volume. However, for cells with a smallsample volume such as the 8-position micro cell (100μl), a temperature sensor blocks the instrumentoptical path. It is then difficult to obtain bothabsorbance and temperature of a samplesimultaneously.Here, a DNA measurement example using the 8-position micro cell with a temperature sensor isoutlined. By using one of the 8-position micro cells asa temperature monitor (Figure 1), the horizontal axisof the temperature course data can be plotted withactual temperatures obtained by the sensor. Thisincreases the temperature accuracy ofmeasurements with the 8-position micro cell.

Measurement System• PAC-743 water-cooled Peltier cell changer• 8-position micro cell block• 8-position micro cell• Silicon cap• Cap pressure fixture• Sensor in cell

Measurement ProgramVWTP-780 Temperature Gradient MeasurementProgram

SamplePoly (dA-dT)-Poly(dA-dT) pH7 KH2PO4-NaOH buffersolution (20 µg/mL)

Measurement Parameters• Number of cells: 7 *1)

• Temperature control sensor: holder• Temperature monitor sensor: cell 8 *2)

• Start condition: Keept within +/- 0.01°C of the targettemperature for 3 seconds

• Data interval: 1°C (20-50°C), 0.1°C (50-70°C), 1°C (70-100°C)

• Ramp rate: 2°C/min• Response: Fast• Measurement wavelength: 260 nm• Reverse temperature measurement: ON

*1) Cell 8 was used only for temperature monitor. Absorbancewas not measured.*2) A silicon cap with a hole illustrated in Figure 2 was used forthe temperature monitor cell. Silicon caps without holes wereused for cells 1 to 7.

Figure 1 Sample Setting

Figure 2Silicon Cap

30


DNA Melting Measurement with the PAC-743/743R Water-cooled Peltier Cell Changer Measurement with a Temperature Sensor in Cell

ResultsMeasurement results are illustrated in Figure 3. Thehorizontal axis of the graph was plotted versus thevalues of the temperature monitor sensor in cell 8.The sample measurement required a total of 5hours; 2.5 hours for the temperature increase dataand 2.5 hours for the reverse temperature course.Table 1 indicates the melting temperature calculatedfrom the temperature course data outlined in Figure3. These results demonstrate melting temperaturesfor the various cells from 61.8 to 62.2˚C (Ave. of6.20˚C), with a standard deviation of 0.13˚C, and acoefficient of variance of 0.20%.

The same measurement was performed whileobtaining sample temperatures using the standardtemperature sensor (the holder sensor) within thePAC-743/743R accessory. Table 2 records the meltingtemperatures calculated from the temperature datausing the holder sensor. Measurement results fromthis experiment provide a melting point varying from63.0 to 63.3˚C that is around one degree higher thanthe temperature obtained when using the sensor inthe sample cell. These results indicate that thetemperature of the holder was around one degreehigher than the actual temperature of the sample.On the other hand, the standard deviation andcoefficient of variance are the same for bothmeasurements. Considering these results, the holdersensor offers sufficient capability to measure areproducible melting temperature for all samplecells. However, to obtain the absolute value forsample melts, a cell temperature sensor is highlyrecommended.

Figure 3 Temperature Course Data

Melting g Temperature (sensor in cell)

Rising Falling

Cell 1 62.0 61.9

Cell 2 62.0 62.1

Cell 3 61.9 62.1

Cell 4 61.9 62.0

Cell 5 61.8 61.9

Cell 6 62.0 61.9

Cell 7 62.2 62.3

Average 62.0 62.0

Std. Dev. 0.13 0.13

C.V. 0.20 0.20

Table 1

Melting g Temperature (holder sensor)

Cell 1 63.3

Cell 2 63.0

Cell 3 63.2

Cell 4 63.0

Cell 5 63.1

Cell 6 63.1

Cell 7 63.1

Average 63.1

Std. Dev. 0.13

C.V. 0.20

Table 2

31


DNA Melting Measurements (3) with the PAC-743/ 743R Water-cooled Peltier Cell Changer “1 mm 10 μL 8-Position Micro Cell Block”

When performing a DNA Melting measurement,most of the samples are only available in extremelysmall amounts. Due to a limited amount of sample, itis essential that the amount used formeasurement/analysis purposes be as little aspossible. However, when sampling a small amount ina high temperature range, volatilization of thesample occurs, frequently complicating the analysisprocess. Additionally, in order to increase theaccuracy of the temperature readings of the samples,one of the 8-Position Micro Cell Block cells is utilizedfor the dedicated temperature monitoring so thatthe temperature in a cell can be used in the meltingdata.

Measurement Systems• PAC-743 Water-cooled Peltier Cell Changer• 1 mm 8-position micro cell block• 1 mm 8-position micro cell• Silicon Cap (attached to 1mm 8-position micro cell

block)• Cap fixture (attached to 1mm 8-position micro cell

block)

Measurement programVWTP-780 [Temperature Ramping Measurement/Melting Analysis] Program

SamplePoly (dA-dT)Poly (dA-dT) pH7KH2PO4-NaOH buffer solution (200μg/mL)

Measurement ParametersStart condition: Keept within +/-0.10˚C of thetemperature setting for 3 secondsData interval: 1˚C (20 - 50˚C), 0.1˚C (50 - 70˚C), 1˚C(70 - 100˚C)Temperature gradient: 2˚C / minResponse: FastWavelength for measurement: 260 nm

Holder sensor

Number of cells: 8 Temperature control sensor: holder Temperature monitor sensor: holder

Internal Cell SensorNumber of cells: 7Temperature control sensor: holderTemperature monitor sensor: cell (8)

ResultsThe melting curves from the results of samplemeasurements with all eight micro cell using the holdersensor are plotted as shown in Figure 2. The timerequired for the measurement was totally 2.5 hours andthe changes of samples volume during the measurementprocess are shown in Figure 3. During the measurement,Nujol was placed on top of the sample cells to preventthe sample from adhering to silicon caps.After completion of the measurement of samples, thesolution levels for each of the 8 cells were higher thanthe upper limit of cells (indicated using a red dottedline), and the decrease in sample volume were almostnot observed by the human eye. In short, by using thesilicon cap and cap fixture, volatilization of samples canbe prevented.

Figure 1 8-position automatic cell changer (Left)

Lid retainer (Center)cell and silicon lid (Right)

Figure 2Melting curve data

[Plotted by using Holder Sensor]

32


DNA Melting Measurements (3) with the PAC-743/ 743R Water-cooled Peltier Cell Changer “1 mm 10 μL 8-Position Micro Cell Block”

In order to enhance the accuracy of the temperature,one of the eight cells (referred to as “cell 8”) wasused exclusively to monitor the sample temperature.Figure 4 shows a result of melting curves using thetemperature readings from internal cell sensor.These temperature values were plotted in thehorizontal axis in Figure 4 using data collected frominternal cell sensor in cell 8. No evaporation wasobserved in this case.

The results of the melting points, calculated from themelting curves data in Figure 2 and 4, are shown in Table1 and 2 below. The results using the holder sensor areshown in Table 1 ranging melting temperature between66.0˚C ~66.2˚C (average 66.1˚C) with standard deviationof 0.08˚C and coefficient of variance of 0.13%. On theother hands, the results using internal cell sensor areshown in Table 2 ranging melting temperatures between63.6˚C ~ 63.8˚C (average 63.7˚C) with standard deviationof 0.08˚C and coefficient of variance of 0.12%. Thisindicates that the temperature using the holder sensorwas approximately 3.5˚C higher than using the internalcell sensor. From these data, it can be concluded that theactual temperature of the holder was 3.5˚C higher thanthe temperature of the original sample in the cell, whileboth the standard deviation and coefficient of variancehad no major differences in their result. In conclusion, tocompare the melting temperatures relatively betweeneach sample, the holder sensor is believed to besufficient, while the internal cell sensor in the sample cellis ideal for measuring the absolute value of meltingtemperatures.

Figure 3 Change in sample volume

before and after measurement [Cell 1 from left]: Upper limit of cell

Figure 5 Change in sample volume

before and after measurement [Cell 1 from left]: Upper limit of cell

Figure 4Melting curve data

[Plotted by using Internal Cell Sensor]

Melting Temperature (Holder Sensor)

Temperature

Cell 1 66.1

Cell 2 66.0

Cell 3 66.0

Cell 4 66.1

Cell 5 66.1

Cell 6 66.0

Cell 7 66.2

Cell 8 66.2

Average 66.1

Std. Dev. 0.08

C.V. 0.13

Melting Temperature (Internal Cell Sensor)

Temperature

Cell 1 63.6

Cell 2 63.6

Cell 3 63.6

Cell 4 63.6

Cell 5 63.7

Cell 6 63.7

Cell 7 63.7

Average 63.7

Std. Dev. 0.08

C.V. 0.12

Table 1 Table 2

33


The Model SAH-769 One-Drop: A Dedicated Accessory for Extremely Small Amounts of Protein and Nucleic Acid Samples

JASCO introduces a new measurement method foran extremely small amount of DNA samples, or anyother sample, by using the model V-630BIOspectrophotometer with the model SAH-769 One-Drop accessory.The SAH-769 measures 5 or 0.6 uL of sampledropped on the disk cell with a 1- or 0.2- mm opticalpath, respectively. The precise optical path is securedby covering the liquid sample with the cover glassintegrated with the unit. The cell and cover glass canbe washed by simply wiping them clean withlaboratory wipers. The simple method for themeasurement of proteins and nucleic acids allowsusers to measure large numbers of samplespromptly. The shorter optical path lengthconfiguration allows measurement of highconcentration samples without further dilution.The V-630BIO, the main unit of the system, utilizes amonochromator with a diffraction grating and adouble-beam optical system to ensure high stabilityfor extremely reliable measurements. The V-630BIOcan be operated by the intelligent Remote Module(iRM) color touch panel control module or utilizingthe cross-platform Spectra Manager softwaredesigned for the Windows operating system. Ineither case, the software includes standard programsfor life science analyses. The [Protein/Nucleic AcidMeasurement] program measures the sampleabsorbance at 260 and 280 nm to calculate theprotein and nucleic acid ratio.The [Temperature Control Measurement] programwith optional Peltier thermostatted cell holders canbe utilized for the DNA melting analysis experiment.

System Configuration• V-630BIO Spectrophotometer for life science field• SAH-769 One-Drop• Dedicated disk cell 1-mm optical path with 5 µL of

sample volume• (Optional disk cell 0.2-mm optical path with 0.6 µL

of sample volume)

Measurement Procedure

1) Drop sample on the cell

2) Close the cover glass and the lid of sample compartment

3) Start sample measurement

4) Cleaning the cell

less than

20 seconds

34



Precision of Quantitative AnalysisSolutions of Calf Thymus DNA (KH2PO4 / NaOHbuffer at pH7) at several concentrations weremeasured by using cells with 1-mm and 0.2-mmoptical paths. The spectra (collected with identicalinstrument parameters) using each cell areillustrated in Figures 1 and 2. The graphs 1 and 2illustrate the calibration curves created using theabsorbance maxima at 260 nm. Both calibrationgraphs demonstrate good linearity.

Figure 1 Absorbance spectra of DNA solution

[optical path: 1 mm]

Measurement ParametersData interval: 0.5 nmMeasurement range: 220 to 315 nmBand width: 1.5 nmResponse: MediumScan Speed: 200 nm/min

Graph 2 Calibration Curve [Optical path : 1 mm]

35

y = 0.0017x - 0.0032Correlation coefficient 1.0000Standard deviation 3.09



Figure 2 Absorbance spectra of DNA solution

[optical path: 0.2 mm]

Graph 3 Calibration Curve [Optical path : 0.2 mm]

Contamination of SamplesThe disk cell and cover glass were wiped clean aftermeasuring the absorbance of a 5-μL high concentrationDNA sample. Then, a 5-μL solvent was measured toevaluate sample cross-contamination of the disk cell. Theresults indicated in Table 3 indicate the wiping is enoughto wash the sample from the cell.

Table 3 Carry over of DNA sample

36

y = 0.0002x + 0.0029Correlation coefficient 0.999880002Standard deviation 18 4930641

After the acquisition of the spectrum, it is possible toperform data manipulation, calculating thetemperature of melting from collected experiment.

This can be done in two ways:• A selected routine within Spectra Analysis allows

to make spectra interpolation in order toextrapolate Tm (Figure 4)

• First derivative of the curve can be performed andthen Tm can be easily determined by evaluatingthe maxima of previously obtained curve (Figure5).


Melting Experiments with V-600 Spectrophotometers Series

Figure 2b Temperature Parameters setting

Figure 2c Temperature Parameters setting

Figure 3 Temperature Melting Main Window

Figure 4Tm Determination from Spectrum Interpolation

Figure 5 Tm Determination from First Derivative Spectrum Analysis

CommentsData array type Linear data arrayHorizontal axis Temperature [C]Vertical axis AbsStart 30°CEnd 97.4 °CData interval 0.2 °CData points 338

Measurement InformationPhotometric Mode AbsBand width(UV/Vis) 2.0 nmResponse MediumMonitor wavelength 258 nmCorrection Off 37


Melting Experiments with V-600 Spectrophotometers Series

V-600 Spectrophotometers Series provide a powerfultool for performing temperature dependentexperiments, when main instrument is coupled withany of the newly developed single or multipleposition Peltier thermostatted cell holders listedbelow (see Figure 1):

• EHCS-760 air cooled single position Peltier cellholder

• ETCS-761 water cooled single position Peltier cellholder

• ETCR-762 water cooled single position Peltier cellholder with thermostatted reference side

• PSC-763 air cooled 6 x 1 multiple position Peltiercell holder

• PAC-743 water cooled 6 x 1 or 8 x 1 multipleposition Peltier cell holder

• PAC-743R water cooled 6 x 1 or 8 x 1 multipleposition Peltier cell holder with thermostattedreference side

• PWC-718 water cooled 8 x 8 multiple positionPeltier cell holder

Spectroscopy Suite Spectra Manager 2 standardfunctionality can be extended by adding severaloptional routines to software main part.In particular, Temperature Programmingmeasurement option allows for the monitoring ofvariations in the absorbance of a sample at fixedwavelength when its temperature is graduallychanged.The Software Parameters Setting (Figure 2a,b,c)allows to setup completely automated temperaturedependent experiments, by performing thermalgradients having complex profiles.It is possible to automatically collect absorbancevariation over an extended temperature interval byselecting the most proper data pitch according to thesample transmittance variation in the selectedregion: for example in regions where variation is verylow, data pitch may be wider, while this has to benarrower in regions of high and rapid variation.

At the same time even temperature change speed canbe selected to be different from one temperatureinterval to the other.

For example, it is possible to setup an experiment likethe following one:

• change temperature from 20°C to 60°C at a rate of5°/min and collect one sample data each 2°

• increase temperature from 60°C to 80°C at a rate of1°/min and collect one sample data each 0.5°

• change temperature from 80°C to 95°C at a rate of2°/min and collect one sample data each 1°

Figure 1 V-630 with PSC-763

Figure 2a Temperature Parameters setting

38

Application UV-0019

Measurement of the reduction reaction of 2,6-dichloroindophenol (DCIP)using the absorption stopped-flow system

IntroductionUsing the absorption stopped-flow measurementsystem, consisting of FS-110 fast scanspectrophotometer and SFS-852 stopped-flowsystem, two to four kinds of liquid sample can bemixed quickly and the changes in absorption spectracan be measured at intervals of 5 msec. This systemallows measurement of rapid enzymatic, catalyticand oxidation-reduction reactions. This applicationnote illustrates an example of determining thereaction rate by using the absorption stopped-flowsystem for the reduction of DCIP, whose color inaqueous solution is changed from blue to colorless asa result of reaction with L-ascorbic acid.

Measurement and analysis system• Absorption stopped-flow measurement system• FS-110 fast scan spectrophotometer• SFS-852 stopped-flow system• Stopped-flow measurement program• Reaction rate calculation program

Samples• 20 mmol/L L-ascorbic acid aqueous solution

(Dissolve the L-ascorbic acid with NaOH/Na2HPO4 aqueous solution and make it to a constant volume. Then, adjust the pH to 7.6.)

• 1 mmol/L DCIP aqueous solution

Measurement conditionsSpectrophotometerOptical pathlength: 2 mmWavelength range: 300 to 800 nmData interval: 1 nmMeasurement interval: 0.010 secMeasurement time: 0 to 3 sec

Stopped-flow systemTime of solution sending: 10 msecMixing ratio: 1:1Volume of solution sending: 50 μLThe measurement is started when the syringe isstopped.

ResultsFigure 2 shows the 3D spectra of the sample. When thereaction is started, the spectrum indicates an absorptionmaximum at approximately 600 nm and the sampleexhibits a blue color. Then, the absorbance in the visiblewavelength range changes to approximately zero within1 sec after starting the measurement, and the sampleturns colorless. Figure 3 shows the time course data forabsorbance at the absorption maximum (604 nm) andthe curve fitted to the data between 0.03 and 2.00 secfor the reaction range, assuming the reaction to be aprimary reaction. The fitted curve is in excellentagreement with the measurement results. A reactionrate of 4.3 sec-1 was calculated.

Figure 1Absorption stopped-flow system

Curve fitting analysis parametersNumber of reaction steps: 1Reaction range: 0.03 to 2.00 secCurve fitting analysis resultReaction rate calculation equation: Y(t) = 0.615066 x exp(-t/0.230571)Baseline equation: Y(t) = 0.0105329Time constant: 0.230967 secRate constant: 4.32963 sec-1Half-life period: 0.160094 sec

Figure 2 3D spectra of reduction of DCIP

Figure 3 Time-course measurement results and fitted curve for sample

absorbance at 604 nm 39

Application UV-0024

Transmission measurement of Volvoxby using MSV-5000 series

IntroductionThe MSV-5000 series microscopicspectrophotometer is capable to analyze microsample/area by both transmission/reflectionmeasurement in the region from UV to Near IR,which can be applied to characterization of microsized sample/area and also impurity analysis.Recently, this type of technology is getting verypopular in bioscience field such as the analysis oflocalized constituents in living cells.Volvox which has localized cellular density due to itsinternal daughter colonies, was measured to obtainabsorption spectra and fixed-wavelength mapping.

System configuration• MSV-5100 UV/Vis Microscopic

Spectrophotometer• MAXY-501 Automatic XYZ Stage

SampleWater containing Volvox was dropped onto amicroscope slide glass and dried. (Figure 1)

Measurement conditionsSpectral bandwidth (UV/Vis): 5.0 nm Scanning speed: 1000 nm/minResponse: Quick Data pitch: 1 nmCassegrain objective: 16 timesAperture: 50 mmφ

Spectrum measurementOne of the daughter colonies inside of mother colony is measured to obtain absorption spectrum.

ResultsMeasured absorption spectrum is shown in Figure 2.Chlorophyll a and chlorophyll b are well known as majorchlorophylls included inland plants and green alga 1),2)

and those absorption spectra are shown in Figure 3.2)

This published data on the literature is measured underacetone solvent condition, and the peak positions ofthose chlorophylls are slightly different depending on thesolvent used, but it’s only approx. 2-7 nm difference inwavelength. Comparing the spectra of chlorophylls withVolvox spectrum, it is assumed that chlorophyll a and bare included in Volvox.

Figure 1 Observation Image of dried Volvox

Figure 2 Absorption spectrum of Volvox

Figure 3 Absorption spectra of chlorophylls2)

(with Acetone solvent)

40

Application UV-0024

Transmission measurement of Volvoxby using MSV-5000 series

Fixed Wavelength Mapping MeasurementFixed wavelength mapping measurement wasexecuted at 672 nm since the peak was observed atthis 672 nm by the absorption spectrummeasurement. Mapping measurement at specificfixed wavelength makes it possible to generate highspeed mapping data.

Measurement conditionsMeasurement Mode: Lattice measurementMeasurement wavelength: 672 nmResponse: FastSpectral Bandwidth: 2 nmCassegrain objective: 16 timesAperture: 30 μmφMeasurement interval: 30 μm

ResultsObservation image and its color-coded diagram bymapping measurement are shown as below and it isconfirmed that the area with higher cellular density inobservation image is exactly in good agreement with thearea of higher absorbance in color-coded diagram.

Figure 5 Color-coded diagram of mapping measurement

Figure 4 Observation Image

Reference Literatures1) Hiroshi Terayama ed., Kisoseikagaku (Revised Edition).Shoukabou, 1970, p.130-131.2) Biochimica et Biophysica Acta (BBA) - Bioenergetics.2011, 1807, 968-976.

41

An average sunscreen spectrum is calculated from the multiplesample spectra and the average submitted to a Windows Excelmacro. The Excel macro calculates the SPF rating and otherrelevant data for the sunscreen sample (Figure 2) which isprinted as a single page report. The results can be used duringformula development or for quality control.

ConclusionsThe Diffey Robson in vitro SPF calculation is a simple, rapid anduniversal analysis method for the determination of SPF valuesof sunscreens using either inorganic and organic screeningcomponents. The versatility of the JASCO V-530spectrophotometer can accomplish the SPF calculation inaddition to providing a full suite of UV-Vis analysis capabilities.

1.) B.L. Diffey, J. Robson, J. Soc. Cosmet. Chem., 40, 127-133,1989.

Automated Sun Protection Factor (SPF) Analysis of Sunscreen using UV-VisDr. Amanda L Jenkins, and Dr. Richard A. Larsen, Jasco Inc.

The increasing awareness of the risks of skin cancer withsun exposure requires that sunscreen products beappropriately tested and labeled. The numerous possiblesunscreen formulations demands that a rigorous analysismethod be available.

IntroductionSunscreens work by either reflecting or absorbing theultraviolet (UV) radiation before it reaches the skin. TheUV-A and UV-B region (400 - 290 nm) is the spectral regionthat must be blocked for effectiveness of the sunscreen.Whether organic or inorganic, the active ingredient thatshields the skin from the sun must be present in sufficientquantity and uniformity to ensure skin protection. Thetraditional method for sunscreen analysis is based on aquantitative analysis of a diluted sample. A series ofstandards based on different concentrations of the activeingredient are measured and a quantitative methoddeveloped based on Beer's Law, A = abc. Where, A = theabsorbance value of an analyte band; a = the absorptivitycoefficient of the analyte band - a constant; b = thepathlength - generally considered a constant; and c =analyte concentration.

Results and DiscussionSpectra of sunscreen formulations were collected over thespectral range 400 - 290 nm with a 0.5 nm data pointresolution on a JASCO V-530 UV-Vis spectrophotometer. Afixed slit width of 2 nm was used with a 'Fast' detector speedand a scan speed of 400 nm/min. Sunscreen formulationswere spread onto a 20 cm2 piece of the 3M Transpore™ tape,the sunscreen covered substrate placed into the samplecompartment and several spectra collected from differentareas. Representative spectra of two different sunscreenformulations are presented as Figure 1.

Application UV-01-04

Figure 1 Sunscreen spectral scans

By contrast, the in vitro method developed by Diffeyand Robson1 uses a UV transparent substrate, 3MTranspore™ surgical tape, to examine the finalsunscreen formulation.No dilution and no standard preparation isnecessary. Simply spread the sunscreen on thesubstrate, place the sample in the V-530spectrophotometer and measure the spectrum.

42

Countermeasure technique against heat island phenomenon- Evaluation of solar reflective paint material -

In recent years, solar reflective material attracts attentionas one of remarkable countermeasure technique againstheat island phenomenon and as sustainable householdbuilding material. These cutting edge sustainable materialshave been evaluated by an independent organization asEnvironmental Technology Verification Project underMinistry of the Environment and its evaluation criterion isin process to be studied and to be established as standardregulation in JIS*. There is solar reflective paint material assustainable material which has been approved by JIS(Japanese Industrial Standard) regulation. As shown inFig.1, solar light has significant energy in near IR region.This solar reflective paint material has specific capability toreflect NIR region light of solar with higher efficiency thanthe other general paint materials. This capability reducesthermal energy on surface to penetrate into buildings,helping to improve an efficiency of air conditioner andcontributes to saving energy.

In this Application Note, some example of evaluation aboutsolar reflective paint material based on JIS K5602 is explained.

System configurationV-670 UV/VIS/NIR SpectrometerISN-723 60 mm dia. Integrating sphere (UV/VIS/NIR)Standard white plate for integrating sphere

Measurement/ Analysis programsVWST-774 Solar/visible light measurement programVWCD-790 Color diagnosis program


SampleTwo aluminum plates were painted by both general waterpaint and solar reflective material paint in two different colorssuch as Gray and Red and dried completely for 7 days.

Figure 1 Standard solar intensity

Figure 2 Conduction of solar thermal energy

Figure 3 Integrating sphere unit

Solar reflective paint material General water paint

Figure 4 Sample surface ( Left : Gray / Right : Red)

43

Countermeasure technique against heat island phenomenon- Evaluation of solar reflective paint material -

1. Spectral measurement resultDiffuse reflectance of samples was measured using[Spectral measurement] program under measurementcondition shown in Table 1. By comparing the resultsshown in Figure 5, it is known that the solar reflectivematerial paint has remarkably higher reflectance in NIRregion than general water paint material.

2. Color Analysis resultBy using the spectra obtained, the color analysis of bothdifferent paint materials was implemented using ColorAnalysis program and the results are shown in Figure 6,indicating the very similar color positions for both Gray andRed on chromaticity diagram. Even colors of two kinds of paintmaterial are quite similar, the characteristics are completelydifferent. Spectral measurement in UV/Vis/NIR region is a veryuseful method to obtain various important information toinvestigate such differences.


3. Calculation result of reflectance of solar lightReflectance of solar light in three different wavelength rangeswas calculated using Solar/visible light measurement programbased on JIS K5602. It is clearly seen that solar reflective paintmaterial has higher reflectance in all wavelength region and inNIR region.

Figure 5 Diffuse reflectance spectra of solar reflective paint material and general water paint

Table 1 - Measurement condition

Measurement range 300 - 2500 nm

Data interval 1 nm

UV/Vis bandwidth 5.0 nm

NIR bandwidth 20.0 nm

Response Fast

Scan speed 1000 nm/min

Correction Base line / Dark

Figure 6 Chromaticity diagram

Gray Red

Solar reflective Water paint Solar reflective Water paint

UV/Vis region 24.09 32.19 21.67 17.60

NIR region 70.39 26.68 64.98 50.61

All wavelength region 44.30 29.75 40.53 31.95

44

Evaluation of sun protection fabrics by using a UPF evaluation system

IntroductionUPF (UltraViolet Protection Factor) is used to indicate theUV shielding performance of sun protection for fabricproducts. The ‘UPF value’ represents the ratio of time forsunburn by UV with and without the protection of thefabric material or product. For example, in the case of skinirradiated by ultraviolet light in 10 minutes with a UPF 50cloth, it takes 500 min (50 (UPF) x 10 min) to obtain thesame amount of sunburn to the skin without using thecloth product. The test method of for UPF calculationswhen using a UV-Vis spectrophotometer is defined inAS/NZS 4399:1996, BS EN 13758-1:2002, the AATCC TestMethod 183:2010, and GBT18830:2009.In this application data, the evaluation of the UPF, UPFrating, UVA transmittance, and UVB transmittance of sunprotection fiber products defined in AS/NZS 4399:1996 byusing the UPF calculation system of a UV/Visspectrophotometer is explained. Also, the fluorescencepropertIes are explained by using a spectrofluorometerbecause fabric products sometimes emit fluorescence byUV light irradiation.

Calculation Method - UPFUPF is calculated by equation (1).

UPF ratingTo calculate the UPF rating measure the transmittancespectrum at more than four different points for the samesample and round down the value, calculated by equation (2),by 5.

Application UV-0036

If the UPF rating is smaller than the minimum of each UPF, thevalue which is calculated by equation (3) are rounded down by5.

if UPF rating is more than 50, UPF rating is defined as 50+

UVA transmittance, UVB transmittanceUVA transmittance is calculated by equation (4) by using theaverage of the transmittance in the range from 315 nm to 410nm.UVB transmittance is calculated by equation (5) by using theaverage of the transmittance in the range from 290 nm to 315nm.

45


SampleT shirt (black) polyester 100%Sports shirt (black) polyester 100%Arm cover (black) rayon 55% polyester 45%

Measurement

3D fluorescence measurement3D fluorescence measurements of the samples wereconducted using a spectrofluorometer to identify thefluorescence property using excitation wavelengths in therange from 290 nm to 400 nm.

Transmittance spectra measurement1. Baseline measurements were performed using a

Spectralon reference tile.2. Transmittance spectra measurements were

performed at 4 different points in the same sample.

* If fluorescence was observed in the 3D fluorescencemeasurement, a Fluorescence Cut Filter Block and aFluorescence Cut Filter (U-330) are required to measuresamples which emit fluorescence in the wavelength rangefrom 450 to 650 nm.

Measurement parametersSpectrofluorometerExcitation bandwidth: 5 nmEmission bandwidth: 5 nmScan speed: 5000 nm/minResponse: 10 msecData interval: 0.5 nmSpectra correction: ON

SpectrophotometerUV/Vis bandwidth: 5.0 nmScan speed: 100 nm/minResponse: 0.96 secData interval: 1 nm

Results of 3D fluorescence measurementsFigure 1 illustrates the 3D fluorescence measurement of the Tshirt. No fluorescence was observed for the excitationwavelengths from 290 to 400 nm. No fluorescence wasobserved for the sports shirt and arm cover samples.

Application UV-0036

Figure 1 3D fluorescence spectra of T shirt

Results of transmittance measurementsFigure 2 shows the transmittance spectrum of each sample.

Figure 2 Transmittance spectrum of each sampleRed line: T shirt

Blue line: Sports shirtGreen line: Arm cover

46


Figure 3 illustrates the UPF measurement software displayand Table 1 provides the analysis results for the samples.As shown in Figure 3, the [UPF measurement] programcan objectively compare the performance of theultraviolet shielding as a result of the numericalcalculation of the UV shielding performance of fabricproducts. Moreover, the program corresponds to variousstandards including BS EN 13758-1:2002, AATCC TestMethod 183:2010, and GBT18830:2009.

Application UV-0036

Figure 3 UPF Measurement Program

Table 1 Analysis result based on AS/NZS 4399:1996

47

Evaluation of the privacy film using an automated absolute reflectance measurement accessory

IntroductionA privacy film which is used for smartphone displays has acharacteristic structure in which clear layers and lightshielding layers are interlaminated. This structureprevents a smartphone display from bystanders ‘peeking’at the screen while the viewing angle depends on theheight and pitch of the light shielding layers in the louverlayers. To evaluate the viewing angle or the transmittanceof the louver layer, an absolute reflectance measurementaccessory is an effective tool. The accessory can be usedto set samples at a specified angle by rotating the sampleto the source incidence and/or detector angles. In thisapplication, the angle dependence of the transmittancespectra of the privacy film for a smartphone is explored.

SamplePrivacy film for smartphones (Figure 2)Specification: View angle 65° Anti-glare processing

Measurement condition• Position: transmittance, asynchronous• Detection angle: 0°• Incidence angle: −60° to 60°• Measurement interval: 2°• Measurement mode: %T• Wavelength range: 380 to 780 nm• Bandwidth: 5 nm• Scan speed: 400 nm/min• Response: 0.96 sec

ResultFigure 5 illustrates the interval data in which spectra wereacquired every 2º from -60º to 60º. Figure 6 outlines thetransmittance spectra at 0º, 10º, 20º, 30º, 40º, 50º, and 60º.Figure 7 provides the angle scanning transmittance data from−60º to 60º at 550 nm. Figures 5 and 6 indicate that the filmabsorbs blue light at less than 400 nm and keeps thetransmissivity constant at more than 400 nm, which meansthat the film displays the light to eyes without a large colorchange. As illustrated in Figure 7, the transmittance isapproximately 5% near the nominal view angle of±32.5º. Thischaracteristic is quite suitable for user privacy. As indicated inthis report, the absolute reflectance measurement system isbest suited to evaluate the incident angle dependenceproperties of various transmittance spectra.

Application UV-0033

Figure 1 Structure of louver layer

Figure 2 Privacy film sample

Figure 3 Measurement image

Figure 5 Interval data of the privacy film

Figure 6 Transmittance spectrum at every 10ºfrom 0º to 60º

Figure 7 Angle dependence of transmittance spectra at 550 nm 48

Thickness Analysis of natural oxide film on microscopic Si pattern

IntroductionThe MSV-5000 series microscopic spectrophotometer isfor transmission/reflection measurement in a widewavelength range from ultraviolet to near-infrared. Itallows the measurement of the area of as small as 10 umdiameter and the built-in high-resolution camera enablesto observe the samples precisely to determine the area tobe measured. This instrument is most suitable to measurethe minute samples or samples having microstructure.This time, the sample on which Si patterns of 35 umwidths are lined up on Ti substrate with 14 um intervalswas measured as a microstructure sample. Actually, thethickness of SiO2 formed upon Si was analyzed from theobtained reflectance spectrum, because Si is easilyoxidized in the air to form thin oxide film of SiO2.

Measurement SystemMSV-5200 Microscopic spectrophotometerVWML-791 [Multi-Layer Analysis] program

Sample: Si and Si oxide film on the Ti substrate

Measurement conditionUV/Vis spectral bandwidth: 5.0 nm NIR spectral bandwidth: 20.0 nmScan speed: 100 nm/min Response: SlowData interval: 0.5 nm Cassegrain objective mirror: 16xIncidence angle: 23ºIN aperture: 10 umφOUT aperture: 10 umφ

Measurement1. Baseline: Al vapor-deposited mirror as a reference is

used for baseline measurement.2. Measurement area: The sample is observed by the

high-resolution camera to determine themeasurement area (Figure 1). The red spot in Figure 1shows the size and position of detected light.

3. Sample measurement: The reflectance spectrum ismeasured.

4. Transforming into absolute reflectance: The absolutereflectance spectrum of the sample is calculated bymultiplying the obtained relative reflectance by theabsolute reflectance of Al vapor-deposited mirror.

AnalysisReflectance(R) is expressed by the equation of refractive indexof the film (ni), extinction coefficient (ki), the angle ofincidence (ϴi), wavelength (λ) and the film thickness (di). Thistime, optical constants of Si and SiO2 are used from theliterature value. and then the film thickness of SiO2 isestimated by using [Multi-Layer Analysis] program by fittingthe calculated reflectance spectrum to the measured one tomake the thickness value reasonable.

Measurement ResultsMeasured absolute reflectance spectrum is shown in Figure 2.MSV-5000 series adopts the confocal optical system, whichenables the measurement eliminating the influence of backside reflection. In the range over 1100 nm where the lightpasses through Si, the spectrum would not be influenced bythe back side reflection.

Application UV-0023

Figure 2 Absolute reflectance spectrum of sample

Figure 1 Observation figure of measurement position

Analysis ResultsThe result of fitting the reflectance spectra using [Multi-LayerAnalysis] program is shown in Figure 3. The error betweenmeasured spectrum and calculated one was within 2% (Figure3) and the film thickness of SiO2 was calculated to be 7.6 nm.

Figure 3 Fitting result of reflectance spectra

Figure 4 The error between measured spectrum and calculated one 49

Luminous Color Measurementby using UV/Vis Spectrophotometer

Lately, products such as LEDs, Organic EL displays,and plasma display panels (PDP) that appliedluminous phenomenon are in widespread use andthe developments in this field are rapidly proceeding.Starting with the blue LED developed in 1993,engineers finally succeeded in developing the whiteLED that is now the mainstream illuminant of themobile phone backlight, flashlight, and so on. Indisplay field, next-generation display with luminousbody, such as the Organic EL displays having superiorperformances on luminous efficacy, flat-screen, andpower consumption and the PDP for large-sized flatTV screen are actively developing.

For these developments, numeric evaluations of colorsand color rendering of the luminous body itself and ofthe manufactured display are required. Here, themethods of evaluating luminous colors and colorrendering by JASCO spectrophotometer are introducedfor the cutting edge technologies of LED, Organic EL, andPDP. Moreover, the method ofevaluating colors of theliquid crystal display (LCD) by the same system is alsointroduced.


Figure 1V-600 series spectrophotometer with the

external source fiber optic interface

Figure 2[Luminous Color Measurement] (Top)

[Luminous Color Analysis] (Bottom)

System Configuration for the Measurement/Analysis

• V-650/660/670 UV/Vis/NIR spectrophotometer• Model ELM-742 External Source Fiber Optic Interface• Calibrated lamp unit• VWLU-788 Luminous Color Measurement/Analysis

Program

50


Main Functions of the[Luminous Color Measurement/Analysis]• Color Calculation Functions

• Tristimulus Values X, Y, Z JIS Z 8701-1999• Chromaticity coordinate (x, y), (u, v), (u’, v’) JIS

Z 8701-1999• Dominant Wavelength λd (Complementary

Wavelength λc), Excitation purity pe JIS Z8701-1999

• Correlated Color Temperature Tcp andDeviation duv JIS Z 8725-1999

• Color Rendering Index Ra, R1 to R15 JIS Z8726-1990

• Classification of the Fluorescent lamps JIS Z9112-1990, 2004

• Pass/Fail Criteria Function

Measuring Method1. Correction Coefficient Spectrum MeasurementSpectrophotometer has different grating efficiency anddetector sensitivity according to the wavelength, so agained spectrum reflects the instrument characteristics.To remove the instrument characteristics, measure thestandard light source having a known emitting pattern inorder to obtain the instrument characteristics(Correction Coefficient Spectrum). Here, the emittingpattern of the standard light source is called as the“Standard light source data”, and a spectrum of thestandard light source measured by thespectrophotometer is a “Standard light sourcespectrum”. “Correction Coefficient Spectrum” can becalculated by dividing “standard light source rawspectrum” by “Standard light source data” (Figure 3).

2. Sample MeasurementMeasure sample after obtaining the CorrectionCoefficient Spectrum. Here, a spectrum measured byspectrophotometer is called as a “Raw spectrum ofsample”, and the “Spectrum Correction” meansremoving the instrument characteristics from thespectrum. The “Spectrum Correction” is executed bydividing the “Raw spectrum of sample” by the“Correction Coefficient Spectrum”.


Figure 3 Calculation of the Correction Coefficient Spectrum

Figure 4 Methods of the Spectrum Correction 51


Example 1 - Color Rendering and Correlated ColorTemperature of IlluminationsWhite LED, fluorescent lamp, and sunlight weremeasured to calculate the Color Rendering Index andthe Correlated Color Temperature. The white LEDsshow relatively favorable Color Rendering Index (Ra)with high Correlated Color Temperature (Tcp). Thecolor of the white LEDs differs according to theirtypes, some of them show deeper blue color.Sunlight shows almost Ra 100 and the fluorescentlamp shows smaller Ra.

Example 2 - Color Calculation of Liquid Crystal DisplayThe screens of a liquid crystal display were measured when it shows white, red, blue, green, yellow, light blue,and deep red. The spectra were calculated by the color calculation program.


Figure 5Emitting spectra of the illuminations (above)

data plotted on the chromaticity diagram (below)

No.Illumination

(Spectrum Color)Tcp(K)

duvλd

(nm)Pe(%)

ReferenceIlluminants

RaClassification

of FL

1White LED1

(Blue)9055 -0.013 469.37 19.28 D9055 86 ------

2White LED2(Light Blue)

8563 0.003 482.54 17.62 D8563 80 ------

3White LED3

(Yellow Green)19359 -0.014 470.37 31.23 D19358 80 ------

4Fluorescent lamp

(Green)4632 0.005 572.78 19.05 P4632 82 N

5Sunlight

(Red)5158 0.003 566.74 9.1 D5158 98 N

Table 1 Correlated Color Temperature and Color Rendering for each illumination

Figure 6Spectra for each screen and (above)

data plotted on the chromaticity diagram (below)

52


The XYZ values were converted into RGB values, andcompared the values with that of display settings.Although the values G and B showed relatively highernumber than set value, we consider the resultsmatch reasonably.

The conversion of the XYZ values into RGB values isexecuted according to the method of Colors & DyeingClub in Nagoya/Osaka.(http://www005.upp.so-net.ne.jp/fumoto/index.htm).


No. Color X Y Z x y Tcp (K)

1 White 18.95 20.56 22.01 0.308 0.3342 6714

2 Red 8.66 4.51 0.19 0.648 0.3376 ------

3 Blue 3.56 1.95 19.37 0.1431 0.0784 ------

4 Green 1.37 2.9 0.67 0.277 0.5868 ------

5 Yellow 15.3 18.54 2.82 0.4173 0.5057 ------

6 Light Blue 10.2 15.96 22.06 0.2115 0.331 ------

7 Deep Red 2.74 1.4 5.1 0.2963 0.1518 ------

Table 2 Color Calculation Result for each color(Two-degree standard observer is applied

to the calculation)

ColorDisplay’ RGB settings RGB converted from XYZ

R G B R G B

White 255 255 255 247 262 256

Red 255 0 0 269 0 0

Blue 0 0 255 0 57 256

Green 0 128 0 0 127 32

Yellow 255 255 0 254 257 30

Light Blue 0 255 255 0 263 259

Deep Red 128 0 128 128 11 139

Table 3 RGB values comparison between display settings and converted XYZ values

53

Bandgap Measurements for Titanium Dioxide Powder

IntroductionThe bandgap energy for semiconductor materials canbe determined from the transmittance or reflectancespectrum using a JASCO spectrophotometer.

Diffuse transmittance spectra for rutile and anatasetitanium dioxide powder were measured. Since fortitanium dioxide, absorption occurs due to forbiddendirect transitions, plotting hν against (hνα)2/3 allowsthe bandgap energy to be determined.

Although, in principle, the bandgap should becalculated using the absorption coefficient, whenonly bandgap absorption occurs, the bandgap energycan be determined directly from the transmittanceor reflectance spectrum. If the sample is a thin filmand an interference pattern is present in thetransmittance or reflectance spectrum, use anabsolute reflectance measurement unit to measurethe spectrum. The interference effect can beeliminated by calculating 100-%T-%R.

Application JE-UV-02-16

VWBG-773 [Band Gap Calculation] program

Diffuse transmittance spectrum for TiO2

Apparatus

• V-770 - UV/Vis/NIR Spectrophotometer• ISN-923 - Integrating sphere unit• PSH-002 – Powder Cell• VWBG-773 - Band Gap Calculation program

54

Reflection Object Color Measurements using Color Diagnosis System

IntroductionThe color diagnosis system for the V-700 series canevaluate the reflection or transmission object colordetermined based on the CIE (CommissionInternationale de l'Éclairage) standard.Reflectance spectra for colored plastic reflectiveplates were measured with or without the specularcomponent using an integrating sphere unit. If thespectrum includes the specular component, it isfound that the lightness is exaggerated and thechroma is diminished. Accordingly, to evaluate thecolor of a mirror or a mirror-like sample surface, it isnecessary to remove the specular component duringmeasurement.


VWCD-960 [Color Evaluation (Color Diagnosis)] program

Colored plastic reflective plates

Reflectance spectrum for blue reflective plate

L*a*b* chromaticity diagram

Standard Standard Name Remarks

JIS Z 8722:2009

Methods of colourmeasurement --Reflecting and transmitting objects.

Reflection object color: the φ60-mm orφ150-mm integrating sphere unitcorresponds to geometric condition d.Transmission object color: the standard cellholder or film holder corresponds togeometric condition e.The φ60-mm or φ150-mm integrating sphereunit corresponds to geometric condition f.

CIE No.15:2004COLORIMETRY, THIRD EDITION

Light Source Standard

D65, AJIS Z 8781-2: 2012, CIE 15: 2004, ISO 11664-2:2007 / JIS Z 8701: 1999, JIS Z 8720: 2000, ASTM E308: 2008

B JIS Z 8701: 1982

CJIS Z 8701: 1999, JIS Z 8720: 2000, CIE 15: 2004, ASTM E308: 2008

D50, D55, D75 CIE 15: 2004 / JIS Z 8720: 2000, ASTM E308: 2008

F1, F3, F4, F5, F6, F8, F9, F10 CIE 15: 2004

F2, F7, F11, F12 CIE 15: 2004, ASTM E308: 2008

55

Reflection Object Color Measurements using Color Diagnosis System


Color Difference Standard

⊿E*ab, ⊿L*, ⊿a*, ⊿b*JIS Z 8730: 2009, CIE 15: 2004, ISO 7724/3: 1984,ASTM D2244: 2011

⊿E*ab, ⊿L*, ⊿C*ab, ⊿H*ab

JIS Z 8730: 2009, CIE 15: 2004, ISO 7724/3: 1984,ASTM D2244: 2011

⊿E00, ⊿E00, ⊿L’, ⊿C’, ⊿H’ JIS Z 8730: 2009, CIE 15: 2004, ASTM D2244: 2011

⊿E94,⊿L*,⊿C*ab, ⊿H*ab JIS Z 8730: 2009, CIE 15: 2004 / ASTM D2244: 2011

⊿ECMC(l:C), ⊿L*, ⊿C*ab, ⊿H*ab

JIS Z 8730: 2009, ISO 105-J03: 2009,ASTM D2244: 2011

⊿E*H, ⊿L, ⊿a, ⊿b JIS Z 8730: 1980, ASTM D2244: 2011

⊿E*uv, ⊿L*, ⊿u*, ⊿v*, ⊿C*uv, ⊿H*uv JIS Z 8730: 2009, CIE 15: 2004, ASTM D2244: 2011

Calculation Items Standard

Tri-stimulus values: XYZJIS Z 8701: 1999, CIE 15:2004, ISO 11664-1: 2007,ASTM E308: 2008

Chromaticity coordinates: x, y JIS Z 8701: 1999, CIE 15: 2004, ASTM E308: 2008

Dominant wavelength: ld, excitation purity: pe

JIS Z 8701: 1999, CIE 15: 2004

Lightness index: L*,Chromaticity coordinates: a*, b*

JIS Z 8781-4: 2013, CIE 15: 2004, ISO 11664-4: 2009, ASTM E308: 2008

ab Chroma: C*ab,ab Hue angle: hab

JIS Z 8781-4: 2013, CIE No.15: 2004, ISO 11664-4: 2008, ASTM E308: 2008

Lightness index: L,chromaticity coordinates: a, b

JIS Z 8730: 2002, ASTM D2244: 2011

Lightness index: L*,chromaticity coordinates: u*, v*

JIS Z 8781-5: 2013, CIE 15: 2004, ISO 11664-5: 2009, ASTM E308: 2008

Hue: H, lightness: V, chroma: C JIS Z 8721: 1993

Whiteness index: WIJIS Z 8715: 1999 / CIE 15: 2004 / ASTM E313: 2010 / ASTM E313: 1996 / ASTM E313: 1973

Tint: Tw

JIS Z 8715: 1999 / CIE 15: 2004 / ASTM E313: 2010 / ASTM E313: 1996

Yellowness: YIJIS K 7105: 1981 / JIS K 7373: 2006, ASTM E313: 2010

Color System Standard

XYZJIS Z 8701: 1999, CIE No.15: 2004, ISO 11664-1: 2007, ASTM E308: 2008

L*a*b*JIS Z 8781-4: 2013, CIE No.15:2004, ISO 11664-5: 2009, ASTM E308: 2008

Lab JIS Z 8730: 2002, ASTM D2244:2011

L*u*v*JIS Z 8781-5: 2013, CIE No.15: 2004, ISO 11664-5: 2009, ASTM E308: 2008

Munsell JIS Z 8721: 1993

Color-matching Function

JIS Z 8781-1: 2012, ISO 11664-1: 2007 / JIS Z 8701: 1999, ASTM E308: 2008 / JIS Z 8701: 1982, CIE 15: 2004

56

Gardner Color Measurement System

IntroductionThe Gardner color measurement system canevaluate the Gardner color as a measure of thedegree of coloration of samples such as boiled oil,varnish, petroleum, liquids used in the production ofchemicals, and substances that become molten whenheated.

This system is compliant with ISO 4630-2:2004 Clearliquids - Estimation of colour by the Gardner colour scale- Part 2: Spectrophotometric method.


V-750 UV/Vis spectrophotometer

The Gardner scale is defined based on thechromaticity of glass or liquid standards numberedfrom 1 for the lightest (light yellow) to 18 for thedarkest (brownish red). By identifying the standardsolution that is closest in color to the samplesolution, the standard color number for the samplecan be evaluated. In this program, the chromaticitycoordinates x and y, and the tristimulus value Y forthe standard samples can be registered b p g yentering them directly or by calculating them fromthe spectrum for the standard sample. The samplespectrum is then measured and its Gardner color iscalculated either from the color difference betweenthe sample and the standard, or by comparing their xand y values.

[Color Evaluation-Gardner Color] program (example)

XYZ color system with standard solution coordinates plotted

57

Hazen Color Measurement System

IntroductionThe Hazen color measurement system can evaluatethe Hazen color of samples such as drying oil,varnish, petroleum, liquids used in the production ofchemicals, and substances that become molten whenheated.

1. The color difference between standard and samplesolutions

2. A calibration curve for the relation between theyellowness index YI and Hazen units for standardsolutions

3. A calibration curve for the relation between thechromaticity coordinates b* and Hazen units

4. A calibration curve for the relation between theabsorbance at a specified wavelength and Hazenunits for standard solutions


V-750 UV/Vis spectrophotometerLSE-701 Long path cell holder 50-mm rectangular cell

The Hazen scale is defined based on the chromaticityof numbered standard solutions with differentconcentrations and yellow hues. By identifying thestandard solution that is closest in color to thesample solution, the standard color number for thesample can be evaluated. In the [Color Evaluation-Hazen Color] program, any of the following methodscan be used to determine the color in Hazen units.

VWHC-977[Color Evaluation–Hazen Color] program

L*a*b* color system with standardsolution coordinates plotted

Calibration curve for YI and Hazen units

This system is compliant with ISO 6271-2:2004 Clearliquids - Estimation of colour by the platinum-cobaltscale-Part 2: spectrophotometric method. In thisprogram, the visual test defined in JIS K 0071-1:1998, ISO6271-1:2004 or ASTM D 1209-05 is applied to the UV/Visspectrophotometer.

58

Color Analysis System for RGB Color Filters

IntroductionThis system measures the transmittance for eachpixel in RGB color filters used for flat panel displays,and analyzes the color. The transmittance spectrumfor the three colors in the filter was measured, andthe calculation results were plotted as a chromaticitydiagram using the [Color Diagnosis Analysis] program


[Color Diagnosis Analysis] program

Chromaticity diagram (XYZ color system)

Observed image

Transmittance spectrum

Model No. Product name Remarks

Instrument MSV-5200-16UV/Vis/NIR

Micro-spectrophotometer

• Wavelength range: 200 to 2700 nm• Supplied with 16x Cassegrain objective, converging mirrors, and manual

stage.

OptionalProgram

MCAN-581[Color Diagnosis

Analysis] program• Dedicated analysis program for Spectra Manager Ver.2• Data in multiple file formats (*.jwa, *.jwb, *.jwd) can be analyzed.

OptionalAccessories

MAXY-501-F Automatic XYZ stage• Optionally installed in factory Movable distance*: X: 76 mm Y: mm, 52

mm, Z: 25 mm. * Varies depending on the magnification of the objective or convergence mirror, or the type of sample.

DescriptionAvailable models are the MSV-5100 (200 to 900 nm) and MSV-5300 (200 to 1600 nm).Using the automatic XYZ stage, mapping, line and multipoint measurements can be performed, in addition to automaticfocusing and multiple-image acquisition. The joystick is an optional extra.

59

Estimation of refractive index of monocrystalline sapphire by polarization measurement using MSV-5000 series

IntroductionThe MSV-5000 series microscopicspectrophotometer is for transmission and reflectionmeasurements of a sample as small as 10 µmφ in awide wavelength range from ultraviolet to near-infrared. The MSV-5000 has a built-in the Glan-Taylorpolarizer as standard and can obtain optical constantsuch as refractive index(n) and extinctioncoefficient(k) by measuring reflectance spectrum ofsmall monocrystalline having birefringence. Thistime, the monocrystalline sapphire(measurementarea size: 50 um in diameter), which has two types ofcrystal axis (c-axis or a-axis) and whose refractiveindex is already known, was measured and thedispersion of the refractive index was calculated.

Measurement systemMSV-5200 UV/Vis/NIR Microscopic spectrophotometer

VWML-791 Multi-layer analysis program

SampleMonocrystalline sapphire

Measurement1. Baseline: Two baselines of Al vapor deposited

mirror were measured with polarizer angle at 0and 90 degrees as a reference.

2. Determination of crystalline axis: Under thecondition of the polarizer angle at 0 degree andwavelength at 550 nm, the sample was rotatedto find the angle of sample where the sampleshowed maximum reflectance. Then c-axis wasdetermined by this angle and as orthogonal of c-axis, a-axis was determined as.

3. Sample Measurement: After determination of c-axis, the reflection spectra were measured atpolarizer angle at 0 and at 90 degreerespectively.

4. Conversion into absolute reflectance: Theabsolute reflectance spectrum of the sample wascalculated by multiplying the obtained relativereflectance by the absolute reflectance spectrumof Al vapor deposited mirror.

AnalysisTwo kinds of calculation methods were used forobtaining the refractive index and the results werecompared.1. Method using [UVVIS K-K Conversion] Program: The

refractive index(n) is expressed by specularreflectance spectrum(R) and phase change(φ)(Equation 1). Since the Kramers-Kronig(K-K) equationcan be applied to specular reflectance spectrum(R)and phase change(φ) (Equation 2), phase change(φ)was calculated by K-K conversion of specularreflectance spectrum (R) and then, the refractiveindex(n) was calculated.

Application UV-0025

2. Method using [Multi-layer Analysis] Program(Application of Fresnel equation): Reflectancespectrum is expressed by the refractive index of theair and the sample(n1, n2), the incident angle(ϴ1)and the reflection angle(ϴ2) (Equation 3). Byapplying this equation, the wavelength dispersion ofthe refractive index was calculated using [Multi-layerAnalysis] Program by fitting the calculatedreflectance spectrum using Equation 3 to themeasured spectrum.

60

Estimation of refractive index of monocrystalline sapphire by polarization measurement using MSV-5000 series

ParametersSpectral bandwidth: 5.0 nmResponse: SlowAccumulation: 3 timesCassegrain objective : 16 timesAngle of polarizer: 0, 90 degreeScan speed: 200 nm/minData interval: 0.1 nmIncident angle: 23 degreeIN aperture: 50 umφOUT aperture: : 50 umφ

Measurement ResultsAbsolute reflectance spectrum of monocrystallinesapphire is shown in Figure 1. Reflectance of ordinarylight (c-axis) is approximately 0.15% higher than thatof extraordinary light (a-axis).

Application UV-0025

Figure 1 Absolute reflectance spectra of monocrystalline sapphire

Figure 2 Wavelength dispersion of refractive index of monocrystalline sapphire

above: by using [UVVis K-K Conversion] Programbelow: by using [Multi-layer Analysis] Program

Analysis ResultsBy using [UVVis K-K Conversion] and [Multi-layerAnalysis] Program, the wavelength dispersion of therefractive index was obtained (Figure 2). Table 1shows the result compared with the literature valueof the reflective index of ordinary light andextraordinary light. The refractive index wasdetermined with precision of two decimal places in asmall area of several tens of microns, by eithercalculating method.

Table 1 Comparison with literature value of the refractive index of monocrystalline sapphire

above: by using [UVVis K-K Conversion] Program below: by using [Multi-layer Analysis] Program

61

Transmittance and Reflectance Measurement System For Minute Lenses

IntroductionSmall type lens is widely used in various products assmart phone , tablet, PC etc. This note show thetransmittance and reflectance measurement for 1-mm-diameter lenses used in mobile-phone cameras.

For samples that refract light such as lenses, thetransmittance can be measured using an integratingsphere. The reflectance can also be measured if theaperture size is sufficiently reduced so that the areabeing analyzed can be considered to be flat.


Integrating sphere for MSV-5200

1-mm-diameter mobile-phone camera lens

Model No. Product name Remarks

Instrument MSV-5200-16UV/Vis/NIR

Micro-spectrophotometer

• Wavelength range: 200 to 2700 nm• Supplied with 16x Cassegrain objective, converging mirrors, and manual

stage.

OptionalAccessories

MISP-552Integrating sphere for

MSV-5200

• Wavelength range: 250 to 2000 nm• Only for the dedicated manual stage for the integrating sphere. Not

applicable for diffuse transmittance or reflectance measurements.• Normal (non-diffuse) reflectance measurements can be performed

using the reflectance optical path.

DescriptionAvailable models are the MSV-5100 (200 to 900 nm) and MSV-5300 (200 to 1600 nm). The type ofintegrating sphere varies depending on the instrument model, and determines the wavelength range that can be used.

62

Evaluation of antireflection filmby using absolute reflectance measurement accessory

63

IntroductionAntireflection film is widely used in many differentproducts of various fields. For example, it is used forwindow and lens in visible region and also for near IRregion laser diode in optical communication purposeand optical materials. The performance ofantireflection film is getting higher recently, which isless than 0.1% reflectance level. And it is the one ofreason for above expanded markets and fields. Thisapplication note proved that JASCO Automatedabsolute reflectance measurement accessory can beapplied to the reflectance measurement with lessthan 0.1%. For this proof statement, severalmeasurement data are shown in below about filmsample which has been designed less than 0.1%reflectance both in visible and near infrared regions.

Sample1) Measurement for linearity• Visible region: KMnO4 solution (66.7, 133.3,

200.0, 266.7 mg/L)• Infrared region: ND filter A, ND filter B2) Reflectance measurement of antireflection film*1)

• AR coat VIS (430 - 600 nm)• AR coat NIR-1 (980 - 1140 nm)• AR coat NIR-2 (1320 - 1600 nm)*1 The base of antireflection film is quartz.

MeasurementMeasurement for linearity1.1 Visible region

(1) Dark measurement is carried out with settingthe masking shield to the cell holder ofAutomated absolute reflectance measurementaccessory.(2) Baseline measurement is carried out withsetting water set into the rectangular cellholder.(3) Sample measurement is carried out withsetting sample into the rectangular cell holder.

Application UV-0040

1.2 Near infrared region(1) Dark measurement is carried out with setting themasking shield to the sample holder of Automatedabsolute reflectance measurement accessory.(2) Baseline measurement is carried out with air.(3) Sample measurement is carried out with NDfilter A set into the sample holder.(4) Sample measurement is carried out with NDfilter B, not parallel to ND filter A.(5) Sample measurement is carried out with settingoff ND filter A.

2.0 Reflectance measurement of antireflection film(1) Dark measurement is carried out with setting themasking shield to the sample holder of Automatedabsolute reflectance measurement accessory.(2) Baseline measurement is carried out with air.(3) Sample measurement is carried out with settingsample to sample holder.

Result(1) Measurement for linearity1.1 Visible regionFigure 1 shows the absorption spectra of KMnO4solution, and Figure 2 shows the calibration curve at 526nm where is peak wavelength. As shown in Figure 2, inabsorbance range from 1 to 4, R2 shows the highcorrelation (more than 0.999). This indicates thatAutomated absolute reflectance measurement accessorycan provide absorbance up to 4, which means thattransmittance and reflectance are obtained up to 0.01%.

Figure 1 Absorbance spectrum KMnO4 solution


64

1.2 Near infrared regionFigure 3 shows the absorption spectrum of ND filter.The spectrum of ND filter A and B agrees in that ofsum of filters. This indicates that Automated absolutereflectance measurement accessory can provideabsorbance up to 4, which means that transmittanceand reflectance are obtained up to 0.01 %.

Application UV-0040

2) Reflectance measurement of antireflection film Figure4 shows the reflectance spectrum of antireflection film invisible region and Figure 5, 6 show in near infraredregion. Table 1 shows the reflectance of the bottompeak. As shown in Figure 4, 5, and 6, V-770/780 allowmeasurement with less than 0.1 % reflectance in thewavelength range from visible to near infrared. V-770and V-780 provides almost similar spectrum in terms ofS/N ratio in the wavelength range from 980 nm to 1140nm. But, in wavelength range from 1320 nm to 1600 nm,V-780 provides the more high-quality spectrum with highS/N ratio due to the difference of installed detector. V-770 equips the PbS detector which has high sensitivity inthe wide wavelength range. On the other hand V-780equips the InGaAs detector which has more highsensitivity. Thus, this means V-770 is suitable formeasurement with wide wavelength range and V-780 issuitable for high sensitivity measurement.

Figure 2 Calibration curve at 526 nm

Figure 3 Absorbance spectra of ND filter

Figure 4 Reflectance spectrum of AR coat VIS

Figure 5 Reflectance spectrum of AR coat in visible region NIR-1


65

Application UV-0040

Measurement parameters

1 Measurement for linearity

1.1 Visible• UV/Vis bandwidth L5.0 nm • UV/Vis response 3.84 sec • Scan speed 100 nm/min• Data interval 1 nm

1.2 Near-infrared• NIR bandwidth 40.0 nm • NIR response 3.84 sec • Scan speed 100 nm/min• Data interval 1 nm

2 Reflectance measurement of antireflection film

AR coat VIS• UV/Vis bandwidth L5.0 nm • UV/Vis response 0.96 sec • Scan speed 200 nm/min• Data interval 0.5 nm• Incident angle 5°• Polarization N-polarized light

AR coat NIR-1/NIR-2• NIR bandwidth 40 nm (V-770)/ 10.0 nm (V-780)• NIR response 3.84 sec• Scan speed 100 nm/min • Data interval 1 nm • Incident angle 5°• Polarization N-polarized light

Figure 6Reflectance spectrum of AR coat in visible region NIR-2

AR coat VIS

Wavelenght [nm] Reflectance [%]

V-770450.5 0.03681

558.0 0.03144

AR coat NIR-1


V-770 1049 0.03655

V-780 1049 0.03655

AR coat NIR-2


V-770 1406 0.03703

V-780 1406 0.04235

Table 1 Reflectance of bottom peak

in reflectance spectrum

Introduction of Haze value measurement system for UV/Vis spectrophotometer

66

Application UV-0042

Measurement MethodWith using integrating sphere, total transmittance (Tt),sample diffusion rate (T4) and scattering rate (T3) whichis required to calibrate the diffusion by instruments aremeasured in the wavelength range from 380 to 780 nm(figure 1). Using equation (4) provides the diffusetransmittance (Td) of sample, and Haze value can becalculated by the ratio between Tt and Td (equation (5)).

IntroductionHaze value is an indicator to show fogged degree ofsamples and recently is often used to evaluate thetransparence and diffuseness of touch panel andsolar cell materials.In this application note, the measurement method ofHaze value and total light transmittance, andmeasurement of Haze value in diffuser panels arereported. Measurement methods on JIS, ISO, andASTM applied to the UV-visible spectrometer in thisapplication.

SampleSix quartz diffuser panels.

Measurement ResultsThe spectrum of total light transmittance (τt) and ofsample diffusion rate (τ4) are shown in figure 3. Thecalculated Haze value is shown in table 1. As shown inthe figure and the table, the difference of fog degreebetween 3 and 4, 5 and 6 are distinguish clearly. Asresult, in this system, the fogged degree of sampleswhich is difficult to be detected by visual observation canbe evaluated by numerical value. So, this system can beeffective to comparison of products or control of quality.

Introduction of Haze value measurement system for UV/Vis spectrophotometer

67

Application UV-0042

Figura 3aTransmittance spectrum of six plate samples -

Sample 1

Figura 3b - Sample 2

Figura 3c - Sample 3

Figura 3d - Sample 4

Figura 3e - Sample 5

Figura 3f - Sample 6

Measurement ConditionBand width 5.0 nm Scan speed 400 nm/minResponse 0.24 sec Data interval 1 nm

Calculation ConditionLight source D65 Color-matching function JIS Z 8781-1View angle 2°

No. T3[%] Tt[%] T4[%] Td[%] Haze [%]

1 0.08 93.06 0.71 0.63 0.7

2 0.08 92.13 4.94 4.86 5.3

3 0.08 90.88 18.7 18.62 20.5

4 0.08 90.7 23.12 23.04 25.4

5 0.08 87.75 69.34 69.27 78.9

6 0.08 85.46 70.56 70.49 82.5

Table 1 Calculation results of Haze value

Example of the measurement of a highly reflective material, a dielectricmulti-layer mirror using absolute reflectance spectroscopy

68

Application UV-0043

The absolute reflectance system can performmeasurement at arbitrary and user-selectable incidentangles; this allows the measurement of dielectric multi-layer mirrors at designated incident angles. As anexample of high reflectance measurement using theJASCO absolute reflectance measurement system, thisapplication note reports the results for a dielectric multi-layer mirror.

SampleDielectric multi-layer mirror

Measurement1. Dark measurement is performed with a light

shielding plate.2. Baseline measurement is performed against air.3. Sample measurement is performed.

The procedure detailed above was repeated three times.

Measurement Condition• Bandwidth 5.0 nm• Scanning speed 20 nm/min• Response 3.84 sec• Data interval 1 nm• Incident angle 10 degree• Polarization p-polarized light

IntroductionDielectric multi-layer mirrors are laminated opticscomprising of a combination of high refractive indexand low refractive index materials. Using theinterference effect, this mirror can provideextremely high reflectance (near 100%) in a specificwavelength range. (Figure 1)

Figure 1 Diagram of the dielectric multi-layer mirror

n1: high refractive indexn2: low refractive index

This type of mirror is widely used in cameras,telescopes and optics for optical communication toreduce loss in light intensity. Therefore, in the qualitycontrol and R&D of dielectric multi-layer mirrors, it isvery important to evaluate the reflectance with veryhigh accuracy. JASCO provides a high performanceabsolute reflectance measurement system using theV-700 series UV/Vis spectrophotometer with double-beam optical system, which has high photometricstability.

Figure 2 Absolute reflectancemeasurement accessory

Example of the measurement of a highly reflective material, a dielectricmulti-layer mirror using absolute reflectance spectroscopy

69

Application UV-0043

Measurement resultFigure 3 shows an overlay of the absolute reflectancespectra of the dielectric multi-layer film mirror, andFigure 4 shows a zoomed view of the same spectra.As shown in the Figure 4, the measurementrepeatability around 100%R is better than 0.1%R,which demonstrates that this system has very highmeasurement repeatability. Table 1 shows thecomparison between the expected values and themeasured values. The difference between theaverage of the measurement value and the expectedvalue is better than 0.15%, which shows that thissystem has high accuracy and good correlation forreflectance measurement close to 100%R.

Figure 3 Absolute reflectance spectra of the dielectric multi-layer mirror

Figure 4 Absolute reflectance spectra of the dielectric multi-layer mirror at around 100%R

Wavelength[nm]

Expectedvalue[%R]

Measurement value [%R]Average

value[%R]

Standardvariation

Variationcoefficient

[%]

Differencebetweenexpected

andaverage

1 2 3

680 99.9914 99.8663 99.8537 99.8407 99.8536 0.013 0.013 –0.1378

700 99.9918 99.8892 99.8943 99.8578 99.8804 0.020 0.020 –0.1114

720 99.9839 99.9094 99.9316 99.8889 99.9100 0.021 0.021 –0.0739

Table 1 Comparison of theoretical and measured values

Measurement reproducibility of Color Diagnosis System

70

Application UV-0044

MeasurementMeasurement was performed using a V-750 UV-Visspectrophotometer with ISV-922 integrating sphere andand VWCD-960 Color diagnosis program. Baselinemeasurement was performed using a standard whitereference plate, then the sample was measured. Bothreference and sample measurement was made with thespecular reflection component included.

To confirm the measurement reproducibility 10 spectrawere collected with the sample removed and replaced inbetween scans to assess the effects of changing themeasurement position.

Measurement Condition• Bandwidth 5.0 nm• Scanning speed 400 nm/min• Response 0.96 sec• Data interval 1 nm

Measurement resultThe reflectance spectra measured for each sample areshown in Figure 3. The right side of Figure 3 showszoomed-in spectra in the wavelength range 740 - 780nm.As shown in each spectrum, the difference betweenmaxima and minima of the photometric value is less than0.15%R, this demonstrates that measurement systemhas high reproducibility.

IntroductionColor is a visually perceived property that is derivedfrom the reflected or transmitted spectrum of lightinteracting with the eye (Figure 1).

It is important to evaluate and obtain measurablefactors describing color for color management duringthe industrial processes used in the manufacture ofproducts.

JASCO has developed various applications that usethe V-700 Series of UV/Vis spectrophotometers forcolor analysis using both reflectance and/ortransmittance spectra of samples. The V-700spectrophotometer Figure 1 Mechanism of colorrecognition by human eye uses a double-beamoptical system, which offers high measurementaccuracy and stability. Because of the highperformance of the V-700 UV/Visspectrophotometer color coordinates (results forcolor calculation) can be determined with highreproducibility.

This application note illustrates the reproducibility ofcolor calculations made using JASCO's proprietarycolor diagnosis system.

SampleColor pellets (blue, red and yellow) (Figure 2)

Figure 1 Mechanism of color recognition by human eye Figure 2 Sample


71

Application UV-0044

Color pellet (Blue)

Color pellet (Red)

Color pellet (Yellow)

Figure 3 Reflectance spectra of color pellets


72

Application UV-0044

Analysis ResultsThe Color Diagnosis Program (Fig.4) is used tocalculate color coordinates from measured spectra.Figure 5 shows the results plotted on XYZ, L*a*b*,Lab and L*u*v* chromaticity diagrams. Thecalculated values are shown in tables 1, 2 and 3. Forthe color coordinates that represent chroma and hue(xy, a*b*, ab, u*v*), the difference between maximaand the minima are; x and y, less than 0.0012, a* andb*, less than 0.31, a and b, less than 0.18, u* and v*,less than 0.50.For the color coordinates that represent brightness(Y, L, L*), the difference between the maxima andminima is less than 0.1.As shown in these results the JASCO Color Diagnosissystem offers high reproducibility.

Figure 4 Color Diagnosis Program

Figure 5a XYZ color system

Figure 5b L*a*b* color system

Figure 5c Lab color system

Figure 5d L*u*v* color system


73

Application UV-0044

Table 1 Color calculation results of color pellet (blue)

Table 2 Color calculation results of color pellet (red)


74

Application UV-0044

Table 3 Color calculation results of color pellet (yellow)

Analysis Conditions• Light source D65 (CIE 15:2004, JIS Z 8781-1:2012, ISO 11664-2:2007)• Color-matching function CIE 15:2004, JIS Z 8701:1982• Viewing angle 2º• Data interval 5 nm

JASCO Europe S.r.l.Via Cadorna, 1 - 23894 Cremella (LC)

[email protected]

www.jasco-europe.com

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DISCLAIMERThe contents of this publication are for reference and illustrative purposes only. Information, descriptions, and specifications in this document are subject tochange without notice and cannot be used from third parts for data comparison and/or performance comparison. JASCO assumes no responsibility and will not beliable for any errors or omissions contained herein or for incidental, consequential damages or losses in connection with the furnishing, performance or use of thismaterial.

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