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Home / Applications / Determination of Fluorescence Excitation/Emission Maxima

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  • Technique

Determination of Fluorescence Excitation/Emission Maxima

By Heather Haffner

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August 25, 2022

Introduction

To determine the most favorable parameters for acquisition of fluorescence spectra, a 3D Fluorescence Measurement Program is included in Spectra Manager  to provide a simple method for establishing the optimum parameters for further experiments.

One of the challenges in performing fluorescence measurement is determining the optimum excitation and emission wavelengths for an unknown sample. With the large amount of information that can be obtained using fluorescence techniques, it is critical to use instrument conditions that offer the best data.  The 3D fluorescence measurement program is used to search for the optimum peak excitation and emission wavelengths for unknown samples. This is done by measuring the emission spectrum of a sample while varying the excitation wavelength or, conversely, measuring the excitation spectrum as the emission wavelength is varied.

Experimental

JASCO FP-6500
Jasco FP-6500 Spectrofluorometer for fluorescence analysis

An FP-6500 Spectrofluorometer with standard 10 mm cuvette holder was used for all analyses. Fluorescence Standard Reference Materials (SRM) were obtained from Starna.

Figure 1. 3-D Spectrum of P-Terphenyl

In Figure 1, the 3D spectra of p-terphenyl, a laser dye and a component of sunscreens, has a maximum excitation of 295 nm and emission of approx. 340 nm.

Quinine is a strongly fluorescence compound that is found in tonic water. In dilute acid solution, there are 2 absorption bands centered at 250 nm and 350 nm and a peak fluorescence emission at 450 nm. After measurement of the 3D spectrum of the tonic water sample, the 3D fluorescence analysis program in Spectra Manager was used to perform an emission search to determine the maximum excitation and emission wavelengths. The results were in good agreement with known literature values.

Figure 2. 3-D Spectrum of Tonic Water
Figure 3. Emission Search Spectra for Tonic Water. Ex. Max: 360 nm, Em. Max: 455 nm

Keywords

p-terphenyl, quinine, spectrofluorometer, emission wavelength, excitation wavelength, FP-6500, FP-8300

Conclusion

Using the 3-D Fluorescence Measurement Program within the Spectra Manager software, it was possible to determine the optimum excitation and emission conditions for various samples.

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.

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About the Author

JASCO Application Note

Determination of Fluorescence Excitation/Emission Maxima

Introduction

To determine the most favorable parameters for acquisition of fluorescence spectra, a 3D Fluorescence Measurement Program is included in Spectra Manager  to provide a simple method for establishing the optimum parameters for further experiments.

One of the challenges in performing fluorescence measurement is determining the optimum excitation and emission wavelengths for an unknown sample. With the large amount of information that can be obtained using fluorescence techniques, it is critical to use instrument conditions that offer the best data.  The 3D fluorescence measurement program is used to search for the optimum peak excitation and emission wavelengths for unknown samples. This is done by measuring the emission spectrum of a sample while varying the excitation wavelength or, conversely, measuring the excitation spectrum as the emission wavelength is varied.

Experimental

JASCO FP-6500
Jasco FP-6500 Spectrofluorometer for fluorescence analysis

An FP-6500 Spectrofluorometer with standard 10 mm cuvette holder was used for all analyses. Fluorescence Standard Reference Materials (SRM) were obtained from Starna.

Figure 1. 3-D Spectrum of P-Terphenyl

In Figure 1, the 3D spectra of p-terphenyl, a laser dye and a component of sunscreens, has a maximum excitation of 295 nm and emission of approx. 340 nm.

Quinine is a strongly fluorescence compound that is found in tonic water. In dilute acid solution, there are 2 absorption bands centered at 250 nm and 350 nm and a peak fluorescence emission at 450 nm. After measurement of the 3D spectrum of the tonic water sample, the 3D fluorescence analysis program in Spectra Manager was used to perform an emission search to determine the maximum excitation and emission wavelengths. The results were in good agreement with known literature values.

Figure 2. 3-D Spectrum of Tonic Water
Figure 3. Emission Search Spectra for Tonic Water. Ex. Max: 360 nm, Em. Max: 455 nm

Conclusion

Using the 3-D Fluorescence Measurement Program within the Spectra Manager software, it was possible to determine the optimum excitation and emission conditions for various samples.

Keywords

p-terphenyl, quinine, spectrofluorometer, emission wavelength, excitation wavelength, FP-6500, FP-8300

This document has been prepared based on information available at the time of publication and is subject to revision without notice. Although the contents are checked with the utmost care, we do not guarantee their accuracy or completeness. JASCO Corporation assumes no responsibility or liability for any loss or damage incurred as a result of the use of any information contained in this document. Copyright and other intellectual property rights in this document remain the property of JASCO Corporation. Please do not attempt to copy, modify, redistribute, or sell etc. in whole or in part without prior written permission.
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