UV-VIS Spectroscopy And Fluorescence Spectroscopy (Part 1 Of 2) - Shimadzu

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C101-E145TALK LETTERVol. 17UV-VIS Spectroscopy and Fluorescence Spectroscopy (Part 1 of 2) ------- 02Applications:Spectrofluorophotometers Used in a Variety of Fields ------- 06Q&A:Is there a way to avoid detecting scattered light andhigher-order light in an emission spectrum? ------- 10

UV Talk LetterUV-VIS Spectroscopy and Fluorescence Spectroscopy (Part 1 of 2)1. IntroductionAll the various things we see with our eyes either generate orabsorb light. The range of light detectable to the human eye iscalled visible light. Using our eyes as detectors, we routinelyperform quantitative and qualitative analyses based on thecolors we observe in our daily lives. Similarly, UV-VISspectroscopy and fluorescence spectroscopy are used toaccurately analyze light in both the visible and ultraviolet lightranges. Both photometric methods measure the samewavelength range, but they differ in the type of samples theycan measure and their detection sensitivity, due to differencesin instrument design. In this two-part series, we will describeboth UV-VIS and fluorescence spectroscopy by comparing theircharacteristics, such as their operating principle, systemcomposition, and advantages and disadvantages. In this firstpart, we will discuss the relationship between light absorptionand fluorescence, the principle of fluorescence, and advantagesof measuring fluorescence over absorption.2. Light Absorption and FluorescenceLight Emission and Absorption Versus ColorThings we can see are either things that emit light, such as theSun, fluorescent lamps, and light-emitting diodes (LEDs), asillustrated in Fig. 1, or things that absorb or reflect a certainportion of the light, giving them the appearance of having aparticular color, as illustrated in Fig. 2.There are a variety of mechanisms for emitting light. Forexample, the Sun emits light due to heat, whereas fluorescentlamps emit visible light when a fluorescent coating inside thelamp is exposed to UV rays. An LED emits light when electricityis applied. Each involves a different method of obtaining energy(stimulus source), which determines the type of emitted light.Table 1 lists some examples of different types of emitted light.Some light emitting items emit a particular color, such as blueLEDs and glow sticks. The color we perceive from emitted lightdepends on the wavelength of light emitted from the emissionlight source. For example, a blue LED emits light near 450 nm,which corresponds to the blue color in Fig. 3. Aspectrofluorophotometer is designed to measure theSunphotoluminescence generated from irradiating a sample withlight, but some models can also measure other types ofluminescence, such as electroluminescence orchemiluminescence.The color we perceive from the absorption, transmission, andreflection of light is related by the color wheel andcomplementary colors shown in Fig. 4. Objects that appear redabsorb light with a wavelength corresponding to thecomplementary color green, which is shown on the opposite sideof the color wheel as red in Fig. 4, so that only light with thewavelength for red is reflected or transmitted through the objectand recognized by our eyes as being red. When a red substanceis measured using a UV-VIS spectrophotometer, an absorptionpeak appears near 530 nm, which corresponds to thewavelength for green in Fig. 3. The colors that can bediscriminated in emitted light depends on the wavelengthsemitted from the light source, but absorption, transmission, andreflection require considering complementary colors.Fluorescent lampFig. 1 Examples of Common Light Emission2LED

UV Talk Le t t e r V o l. 1 7Fig. 2 Absorption and Reflectance Versus ColorTable 1 Types of Light EmissionTypes of Light Emission(Luminescence)Stimulus Source(How Energy Is Obtained)ExamplesThermal RadiationHeatThe Sun, light bulbsPhotoluminescence(fluorescence, phosphorescence)PhotoirradiationFluorescent lampElectroluminescenceVoltageLight-emitting diodes (LEDs) and organic electroluminescent displaysChemiluminescenceChemical reactionGlow sticks (peroxyoxalate chemiluminescence), luminol reactionBioluminescenceChemical reaction(oxidative enzymes, most of which are considered chemiluminescence)Fireflies, footballfish650700Near gth 400Complementarycolors(nm)Fig. 3 Relationship Between Wavelength and Color of LightLight Absorption and FluorescenceFig. 4 Color WheelExcited stateNon-radiative transitionFluorescenceLightLight absorptionWhen light is shined on a particular substance, the substanceabsorbs specific wavelengths of light that are determined by itsmolecular structure, as shown in Fig. 5. This indicates thatelectrons in the ground state (stable state) of the molecule haveabsorbed the energy from the light and transitioned to an excitedstate (a higher-energy unstable state). Then the electrons thathave transitioned to an excited state lose that energy due torelease of heat, collision with other molecules, or other factors,and return to its ground state. That process, referred to asnon-radiative transition, can involve releasing the previouslyabsorbed light energy as light again when they transition to itsground state. In the case of a UV-VIS spectrophotometer, thelight absorption depends on the presence of a particularsubstance and an absorption spectrum can be measured bydetecting the substance. In contrast, a spectrofluorophotometerproduces an emission spectrum by shining light on a substanceand measuring the resulting light emission from the substance.Ground stateFig. 5 Diagram of Energy Levels3

UV-VIS Spectroscopy and Fluorescence Spectroscopy (Part 1 of 2)Absorption, Fluorescence, and Energy LevelThis section describes an absorption spectrum and an emissionspectrum based on Fig. 6, which is a more detail diagram of Fig. 5.In Fig. 6, the electron ground state of a molecular is represented byS0 and the excited state by S1. Each state involves much smallervibrational energy levels than electron energy. Those levels arerepresented by V0 j (where j 0, 1, 2, .) at the electron groundstate and by V1 j at the electron excited state. It shows energy onthe vertical axis and the distance between atoms on the horizontalaxis.At room temperature, a molecule exists at the lowest energy levelV0 0 at S0. When the molecule absorbs light with a wavelength(frequency) corresponding to an energy difference between S0 andS1, then electrons in it transition as indicated in (1) of Fig. 6. AUV-VIS spectrophotometer measures this transition as an absorptionspectrum and a spectrofluorophotometer as an excitation spectrum.The time required for an electron to transition from S0 to S1 is about10-15 seconds. Therefore, an electron transitions from S0 to S1 beforeatom nuclei positions can change within molecule due to stretchingvibration or other factors. Consequently, the positions of atomicnuclei in the molecule do not change immediately before or after theelectron transition. This rule is referred to as the Franck-Condonprinciple. Since the electron localization within the molecule at S1 isdifferent from that at S0, the atomic nuclei positions at eachvibrational energy level of S1 are not same as those of S0. Whileelectrons transition to S1 keeping the atomic nuclei positions at V0 0at S0, their positions are unstable in the excited state due to highvibrational energy. This high-energy state is referred to as theFranck-Condon state. In order to achieve the stable atomic nuclei40000.0(2) Non-radiative transitionV1 4V1 3V1 2V1 1(1) Light absorption(excitation)V1 0Energy(3) FluorescenceV0 4V0 3V0 2Fluorescence intensityElectron excitedstate S1positions with the lowest energy at V1 0, the molecule existing atthe Franck-Condon state will release any excess energy asvibrational energy by moving nuclei positions, so that a non-radiativetransition to V1 0 occurs without any light emission. When theelectron returns from V1 0 back to S0 ((3) in Fig. 6), excess energyis emitted as light. That light is called fluorescence. Compared to theexcitation process (1) in Fig. 6, the emission process (3) has anarrower energy band. That is why the emission spectrum appears inthe longer wavelength region than the absorption (excitation)spectrum.Fig. 7 shows the excitation and emission spectra of anthracene incyclohexane measured using a spectrofluorophotometer. The labelssuch as "0 1" shown in Fig. 7 indicate the transition from V0 0 at S0to V1 1 at S1. It shows how the intervals between peaks in theexcitation spectrum depend on the difference in energy between thevibrational energy levels of S1. Naturally, the intervals between peaksin the absorption spectrum measured using a UV-VISspectrophotometer also reflects the energy differences betweenvibrational energy levels in the excited state, as shown in Fig. 8.The intervals between peaks in the emission spectrum depend onthe difference in energy between the vibrational energy levels at S0,but those energy differences cannot be confirmed using the UV-VISspectrophotometer. In addition, a 0-0 band appears in thewavelength region where the excitation and emission spectraoverlap. When the intervals between vibrational energy levels in theexcited and ground states are similar, the excitation and emissionspectra are mirror reflections of each other, centered at the 0-0band.30000.0Excitationspectrum 0 220000.010000.00 10–01 00 3Emissionspectrum2 00 43 00.0280.0300.0350.0400.0450.0Fig. 7 Excitation and Emission Spectra of Anthracene in CyclohexaneV0 1Electron groundstate S0V0 00.600Distance between atoms0.400AbsFig. 6 Absorption, Fluorescence, and Energy Level0.2000.000280.00300.00350.00400.00Wavelength (nm)Fig. 8 Absorption Spectrum of Anthracene in CyclohexaneMeasured Using a UV-VIS Spectrophotometer4480.0Wavelength (nm)

UV Talk Le t t e r V o l. 1 7Absorption, Excitation and Emission SpectraA UV-VIS spectrophotometer measures an absorption spectrum(transmission spectrum) based on the decrease in light intensitythat occurs when the monochromatic light incident on thesample is absorbed by the sample while it is transmitted throughthe sample. In other words, a UV-VIS spectrophotometermeasures the decrease of light from the instrument light source.In contrast, there are mainly two measurement methods used fora spectrofluorophotometer. One measurement methoddetermines an emission spectrum by dispersing fluorescenceemitted from the sample in response to a fixed excitation light.The other method measures an excitation spectrum at a fixedemission wavelength observed in response to a continuouslyvaried excitation light. In other words, as mentioned above, aspectrofluorophotometer measures the light emitted from thesample. Fig. 9 shows an illustration of the measurement processof each spectral measurement method.By measuring the wavelength and intensity of fluorescenceemitted from a sample, an emission spectrum show whichemission wavelengths of light the sample is emitting mostintensely. In contrast, an excitation spectrum can measure whichexcitation wavelengths of light result in emitting fluorescencemost intensely (optimal excitation wavelength). In most cases,wavelengths with high absorption also result in high excitationand high intensity fluorescence. Similarly, wavelengths with lowabsorption result in minimal excitation and low fluorescenceintensity.Incident lightTransmitted lightSampleAbsorption spectrumExcitation lightFluorescent lightExcitation lightFluorescent lightSampleSampleEmission spectrumExcitation spectrumFig. 9 Illustration of Absorption, Emission and Excitation SpectraDifferences in Measurement SelectivityAn absorption spectrum obtained from a UV-VISspectrophotometer is only related to the light absorbed by amolecule, whereas an emission spectrum obtained from aspectrofluorophotometer is related to two factors, excitation andemission. Therefore, even if molecules A and B both appear atsimilar wavelength regions in the absorption spectrum, as longas there is a difference between the minimum vibrational energylevels of their electron excitation states, then they will appear indifferent wavelength regions in the emission spectrum.Consequently, fluorescence spectroscopy is more likely thanUV-VIS spectroscopy to obtain spectral measurements andintensity values less affected by other components.Precautions for Fluorescence SpectroscopyAbsorption is observed when a sample is exposed to light witha wavelength corresponding to the energy difference betweenground and excited states. Therefore, a UV-VISspectrophotometer, which measures incident and transmittedlight, is able to measure a spectrum for all molecules thatabsorb UV-VIS region light. In contrast, because not allmolecules emit fluorescence, transitions are non-radiative whenexcess energy is released as heat. The ability to measure amolecule with a spectrofluorophotometer is determined bywhether energy deactivation is caused by a non-radiativeprocess or light emission. Even if a molecule emits fluorescence,it releases some of its excess energy from the excited state asheat, rather than releasing it all as light when it returns to theground state.3. SummaryIn this first part, we discussed the relationship between light absorption and fluorescence, the principle of fluorescence, characteristicsof fluorescence as compared to absorption and precautions for fluorescence. In the second part, we will discuss the fluorescencemeasurement in more depth, while also describing the construction of a spectrofluorophotometer.Global Application Development Center, Analytical & Measuring Instruments DivisionTakahiro TajimaKazuki Sobue5

UV Talk Letter ApplicationSpectrofluorophotometers Used in a Variety of FieldsA spectrofluorophotometer can be used to analyze samples in a wide variety of fields. This article gives variousexamples of using the RF-6000 spectrofluorophotometer for analysis in a variety of fields.1. Chemistry[Analysis of Coumarin in Diesel Oil]In Japan, diesel oil is subject to local taxes (diesel oil deliverytax), but not kerosene or class-A fuel oils. Consequently, somevendors have abused the system to avoid taxes by mixinguntaxed kerosene or class-A fuel oil with diesel oil. To identifysuch tax evasion, 1 ppm of coumarin has been added tokerosene and class-A fuel oil and a method for identifying thepresence of coumarin in diesel oil has been specified in aJapan Petroleum Institute standard (JPI-5S-71-2010). Whencoumarin is isomerized, it forms trans-o-hydroxycinnamic acid,which emits fluorescence. Therefore, the quantity of coumarincan be determined based on measuring the intensity of itsfluorescence.Emission spectra for different concentrations of standardcoumarin solutions are shown in Fig. 1 and the calibration curvein Fig. 2. The squared correlation coefficient of the calibrationcurve, r 2, was 0.99965, which indicates good quantitativeaccuracy. For more details, refer to Shimadzu Application Newsbulletin A494 Simplified Measurement of Coumarin in Diesel Oil.150000.0Calibration curveFluorescence intensityFluorescence 550.0600.0nm630.0100000.050000.00.00.0y 1.10881e 005x 0.000000r2 0.99965Fig. 1 Emission Spectra for Different Concentrations ofStandard Coumarin Solutions0.51.01.2Concentration (mg/L)Fig. 2 Calibration Curve for Standard Coumarin Solutions2. Foods[Classifying and Identifying Types of Milk]Three-dimensional spectra of commercial non-fat, low-fat, and high-fat milk products are shown in Fig. 3. This indicates the possibility ofusing three-dimensional spectra to discriminate between the types of milk.Non-fat milkLow-fat milkHigh-fat milkFig. 3 Three-Dimensional Fluorescence Spectra of Commercial Non-Fat, Low-Fat, and High-Fat Milk6

UV Talk Le t t e r V o l. 1 73. Electrical/Electronics[Evaluating the Fluorescence Quantum Efficiency of Solid-State Semiconductor Materials]The ratio of photons from excitation light that are absorbedversus emitted as fluorescence by a fluorescent substance isreferred to as the fluorescence quantum yield or quantumefficiency. These values serve as important indices forevaluating the luminous efficiency or luminescence intensity offluorescent substances. By using an integrating sphere unitaccessory with the RF-6000 spectrofluorophotometer, thequantum efficiency of fluorescent substances can bedetermined easily.Fig. 4 shows an example of using an integrating sphere unit tomeasure the fluorescence quantum efficiency of a solid-statesemiconductor material (tris(8-hydroxyquinolinato)aluminum)used for light-emitting layers of organic electroluminescentdevices. In Fig. 4, "AF" refers to the ratio of the number ofphotons irradiated to the sample to the number of photonsabsorbed by the sample, "QEin" refers to the quantumefficiency, and "QEex" the ratio of the number of photonsirradiated to the sample to the number of photons emitted asfluorescence.Using LabSolutions RF software allowed fluorescence quantumefficiency (fluorescence quantum yield) to be calculatedinstantly using intuitive operations.Fig. 4 Fluorescence Quantum Efficiency of Solid-State Semiconductor Material4. Environmental Fields[Measuring Oils in Water by ASTM D5412]* Synchronous fluorescence spectroscopy simultaneously scans both theexcitation and emission monochromators keeping a given wavelength intervalbetween them. For more details, refer to Shimadzu Application News bulletinA500 Separation Analysis by Simultaneous Fluorescence Spectroscopy.80.0Fluorescence intensityThe American Society for Testing Materials (ASTM) has issuedASTM D5412 test standards for analyzing the quantity ofpolycyclic aromatic hydrocarbons classified as carcinogenicsubstances in oils contained in water. Fig. 5 shows an emissionspectrum for a mixture of five kinds of polycyclic aromatichydrocarbons. Normal measurements only provide a spectrumfor the mixture of five kinds of hydrocarbons, but by usingsynchronous fluorescence spectroscopy*, each component canbe confirmed, as shown in Fig. 6, even if the sample containsmultiple components.40.00.0350.0400.0nm450.0Fig. 5 Emission Spectrum from Mixture of Five PolycyclicAromatic Hydrocarbons (Ex. 300 nm)7

UV Talk Letter g. 6 Left : Synchronous Fluorescence Spectrum of Benzo[a]pyreneRight: Synchronous Fluorescence Spectrum from Mixture of Five Polycyclic Aromatic Hydrocarbons(both offset by 6 nm)5. Life Sciences[Measuring Fluorescent Dyes for DNA Detection]Fluorescent probes are used in virus test kits. These probesform a complementary bond with the DNA of the target virus.The presence of the virus can be determined by measuring theemission intensity from the fluorescence probe bonded to theDNA by complementary bond. Fig. 7 shows an example ofthree-dimensional spectra from a fluorescence probe dye. EvenFluorescence wavelength (nm) / Excitation wavelength (nm)if a small sample quantity is diluted, a spectrofluorophotometercan be used to confirm the presence of the virus with goodsensitivity. For more details, refer to Shimadzu Application Newsbulletin A490 Three-Dimensional Spectra Measurement ofFluorescent Probes used for DNA Detection.Fluorescence wavelength (nm) / Excitation wavelength (nm)Fig. 7 Three-Dimensional Spectra of Two Fluorescent Probes8

UV Talk Le t t e r V o l. 1 76. Pharmaceuticals[Measuring Duloxetine Hydrochloride]Duloxetine hydrochloride is a compound listed in the USP as an antidepressant. Spectra and a calibration curve from duloxetinehydrochloride are shown in Fig. 8. They show that the RF-6000 spectrofluorophotometer can be used to accurately measure duloxetinehydrochloride down to very low concentration levels (lower limit of quantitation of 0.0007 μg/mL and lower limit of detection of 0.0002 0000.0100.2000.4000.6000.800Concentration (μg/mL)Fig. 8 Left : Emission Spectra of Duloxetine HydrochlorideRight: Calibration Curve7. SummaryA spectrofluorophotometer offers higher sensitivity than aUV-VIS spectrophotometer and the RF-6000 in particular canprovide real-time spectral correction using the instrumentfunction. (Refer to the next issue for more information about theinstrument function.) This provides true spectra that are notbiased by instrument functions. The RF-6000 is also capable ofmeasuring long wavelengths up to 900 nm, which enable highsensitivity measurements over a wide range of wavelengths.Furthermore, due to its high-speed three-dimensional spectrameasurement capability, it can quickly determine excitation andemission wavelengths for developing new materials.Thus, a spectrofluorophotometer offers a variety of advantagesand serves as an indispensable tool in many different fields.Global Application Development Center, Analytical & Measuring Instruments DivisionAkara Hashimoto9

Is there a way to avoid detecting scattered light andhigher-order light in an emission spectrum?Sometimes scattered light or higher-order light is detected when measuring an emission spectrum. However,scattered light and higher-order light can be removed using a filter. Fig. 1 shows three-dimensional spectra ofRhodamine B in ethanol measured using a spectrofluorophotometer. Excitation wavelength is plotted on the verticalaxis and emission wavelength on the horizontal axis. Because the excitation and emission wavelengths are thesame in the diagonal region enclosed in a yellow line, it indicates the light scattered from the excitation light. Thediagonal area enclosed in a red line indicates Raman scattered light from the solvent and the region enclosed inthe white line indicates the area with the emission spectrum for Rhodamine B. The areas indicated with green linesindicate higher order excitation light, identified by being two and three multiples of the excitation wavelength(2nd-order starting from 500 nm and 3rd order from 750 nm). The blue line indicates light with half the wavelengthof the excitation light that was emitted at the same time as the excitation light. The higher order light is acharacteristic of the diffraction grating and, therefore, can be removed by filtering. For more information abouthigher order light from the diffraction grating, refer to UV Talk Letter Vol. th (nm)500800Emissionwavelength (nm)Fig. 1 Three-dimensional Spectra of Rhodamine B in 00Fig. 2 Using Y-50 Filter10800520540560580600620Fig. 3 Smaller Measurement Area640

UV Talk Le t t e r V o l. 1 7When selecting a filter for removing scattered light or higher-order light, it is important to be careful of both the emission wavelengthregion of the sample and the wavelength of higher-order light. In Fig. 1, the emission spectrum appears between 550 and 700 nm.Therefore, one filter criterion is that it must be able to pass wavelengths longer than 550 nm. Based on the second-order lightoverlapping with the emission spectrum, indicated by the green line in Fig. 1, a second filter criterion is the ability to blockwavelengths between 275 and 350 nm, which is half wavelengths of the emission spectrum. Based on these two criteria, the Y-50 filterwas selected from the filter set (P/N S204-04691) for Table 1. The spectrum measured using the Y-50 filter, as shown in Fig. 2, showsthat the spectrum could be measured without detecting the scattered light with wavelengths shorter than 500 nm and the higher-orderlight in the region between 500 and 850 nm. However, it is necessary to beware that using a filter decreases the fluorescence intensityand prevents detecting a signal from the wavelength region that was filtered out. On the other hand, in cases where the higher-orderlight does not overlap with the emission spectrum of the sample, or where the region in which higher-order light overlaps with theemission spectrum does not need to be measured, it is also possible to avoid its detection by specifying a narrower measurementwavelength range that does not include the scattered or higher-order light, rather than using a filter, as shown in Fig. 3.Table 1 lists the properties of the high-pass filters included in the filter set. As an example, the wavelength properties of the Y-50 filterare shown in Fig. 4. The number in the filter names typically indicates the wavelength where transmittance is 50 %. The filters alsoblock second and third-order multiples of the excitation wavelengths. Use Table 1 as a reference for selecting a filter appropriate forthe given measurement.Table 1 Filters and Corresponding WavelengthsFilterExcitation Wavelengths FilteredEmission Wavelength PassedIHU-310200 to 310 nmOver 310 nmL - 42200 to 420 nmOver 420 nmY-50200 to 500 nmOver 500 nmO-56200 to 560 nmOver 560 nmR-60200 to 600 nmOver 600 900.0Fig. 4 Wavelength Properties for Y-50 Filter11

For Research Use Only. Not for use in diagnostic procedures.This publication may contain references to products that are not available in your country. Please contact us to check the availability of theseproducts in your country.Company names, products/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation, itssubsidiaries or its affiliates, whether or not they are used with trademark symbol “TM” or “ ”.Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or notthey are used with trademark symbol “TM” or “ ”.Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own.www.shimadzu.com/an/The contents of this publication are provided to you “as is” without warranty of any kind, and are subject to change without notice. Shimadzudoes not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. Shimadzu Corporation, 2016

spectroscopy and fluorescence spectroscopy are used to accurately analyze light in both the visible and ultraviolet light ranges. Both photometric methods measure the same wavelength range, but they differ in the type of samples they UV-VIS Spectroscopy and Fluorescence Spectroscopy (Part 1 of 2) Fig. 1 Examples of Common Light Emission

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