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Home / Applications / Eliminating Fluorescence Artifacts from the Diffuse Reflectance Measurement of Sodium Salicylate

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Eliminating Fluorescence Artifacts from the Diffuse Reflectance Measurement of Sodium Salicylate

By John Burchell

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August 15, 2022

Introduction

Diffuse reflectance measurement with integrating sphere
V-670 UV-Visible/NIR spectrophotometer

A fluorescent powder may be evaluated by its fluorescence spectrum, excitation spectrum, external/internal quantum efficiency and luminescent color.  In addition, a diffuse reflectance spectrum may be required for evaluating its absorption spectrum.  However, when making diffuse reflectance measurement of fluorescent materials using a UV-visible spectrophotometer with integrating sphere, since the scattered light and sample fluorescence cannot be separated, the sum of these will combine to provide the result of a reflectance measurement.  Therefore, to obtain the true reflectance, this fluorescence artifact should be corrected for.

In this application, the procedure to eliminate a fluorescence artifact will be demonstrated using sodium salicylate as the sample (a typical powdered sample that exhibits fluorescence).

Experimental

Instrument

  • V-650 UV-visible spectrophotometer
  • ISV-722 60 mm UV-visible integrating sphere
  • Powder sample holder
  • Reference white plate
  • L42 band pass filter

Measurement

1). In order to reduce the artifact caused by fluorescence, an L42 band pass filter was placed in front of the detector so that the fluorescence is rejected and the scattered light from the irradiation light is transmitted.

2) In single beam mode, spectra were measured of a standard white plate and sample without using the L42 filter.

3). The same single beam spectra were measured while using the L42 filter.

The transmittance of the L42 filter is calculated as the ratio of two spectra of the standard white plate measured with and without filter Fig. 1 (a). The single beam spectra of sodium salicylate measured with and without filter are shown in Fig. 1 (b). The L42 band pass filter has 50% transmission at 420 nm, and cuts wavelengths shorter than 420 nm, it is evident that the transmittance in the range shorter than 420 nm was significantly reduced when measured with the filter.  Also, the spectrum of sodium salicylate measured without the filter indicates that most of the signal intensity at wavelengths shorter than 370 nm was from fluorescence.

 

Single Beam Spectra
Figure 1. Single Beam Spectra

The fluorescence spectra of sodium salicylate measured with and without L42 filter are shown in Fig. 2. The peak measured with filter (light blue) was observed as fluorescence and the peak without filter (pink), as total fluorescence, the ratio of the peak area was calculated, this is used as a factor for obtaining the total fluorescence from the fluorescence. The calculated ratio was 1.0556, by multiplying the single beam spectrum of sodium salicylate measured with filter by this factor (light blue; Fig. 1, b), the total fluorescence was obtained. Then, the true diffuse reflectance component was obtained by the subtracting the total fluorescence from the single beam spectrum of sodium salicylate measured without filter (pink; Fig. 1, b).

Fluorescence spectra of sodium salicylate
Figure 2. Fluorescence spectra of sodium salicylate

The true diffuse reflectance component of sodium salicylate is shown in Fig. 3 together with the diffusive reflectance component of the standard white plate obtained using the same procedure.  Since the component of the standard white plate can be considered as the 100% line, the diffuse reflectance spectrum of sodium salicylate (Fig. 4) was obtained from the ratio of the two spectra shown in Fig. 3.

NOTE: The cut-off wavelength of L42 filter is 410 nm.  Therefore, in this example the correction procedure can only be applied to a wavelength range shorter than 410 nm.

 

Diffuse reflectance component (left), Diffuse reflectance spectrum of sodium salicylate (right)
Figure 3. Diffuse reflectance component (left), Figure 4. Diffuse reflectance spectrum of sodium salicylate (right)
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

John Burchell is a seasoned JASCO veteran adept with chromatography and spectroscopy products. He is currently the business development manager for both instruments.

JASCO Application Note

Eliminating Fluorescence Artifacts from the Diffuse Reflectance Measurement of Sodium Salicylate

Introduction

Diffuse reflectance measurement with integrating sphere
V-670 UV-Visible/NIR spectrophotometer

A fluorescent powder may be evaluated by its fluorescence spectrum, excitation spectrum, external/internal quantum efficiency and luminescent color.  In addition, a diffuse reflectance spectrum may be required for evaluating its absorption spectrum.  However, when making diffuse reflectance measurement of fluorescent materials using a UV-visible spectrophotometer with integrating sphere, since the scattered light and sample fluorescence cannot be separated, the sum of these will combine to provide the result of a reflectance measurement.  Therefore, to obtain the true reflectance, this fluorescence artifact should be corrected for.

In this application, the procedure to eliminate a fluorescence artifact will be demonstrated using sodium salicylate as the sample (a typical powdered sample that exhibits fluorescence).

Experimental

Instrument

  • V-650 UV-visible spectrophotometer
  • ISV-722 60 mm UV-visible integrating sphere
  • Powder sample holder
  • Reference white plate
  • L42 band pass filter

Measurement

1). In order to reduce the artifact caused by fluorescence, an L42 band pass filter was placed in front of the detector so that the fluorescence is rejected and the scattered light from the irradiation light is transmitted.

2) In single beam mode, spectra were measured of a standard white plate and sample without using the L42 filter.

3). The same single beam spectra were measured while using the L42 filter.

The transmittance of the L42 filter is calculated as the ratio of two spectra of the standard white plate measured with and without filter Fig. 1 (a). The single beam spectra of sodium salicylate measured with and without filter are shown in Fig. 1 (b). The L42 band pass filter has 50% transmission at 420 nm, and cuts wavelengths shorter than 420 nm, it is evident that the transmittance in the range shorter than 420 nm was significantly reduced when measured with the filter.  Also, the spectrum of sodium salicylate measured without the filter indicates that most of the signal intensity at wavelengths shorter than 370 nm was from fluorescence.

 

Single Beam Spectra
Figure 1. Single Beam Spectra

The fluorescence spectra of sodium salicylate measured with and without L42 filter are shown in Fig. 2. The peak measured with filter (light blue) was observed as fluorescence and the peak without filter (pink), as total fluorescence, the ratio of the peak area was calculated, this is used as a factor for obtaining the total fluorescence from the fluorescence. The calculated ratio was 1.0556, by multiplying the single beam spectrum of sodium salicylate measured with filter by this factor (light blue; Fig. 1, b), the total fluorescence was obtained. Then, the true diffuse reflectance component was obtained by the subtracting the total fluorescence from the single beam spectrum of sodium salicylate measured without filter (pink; Fig. 1, b).

Fluorescence spectra of sodium salicylate
Figure 2. Fluorescence spectra of sodium salicylate

The true diffuse reflectance component of sodium salicylate is shown in Fig. 3 together with the diffusive reflectance component of the standard white plate obtained using the same procedure.  Since the component of the standard white plate can be considered as the 100% line, the diffuse reflectance spectrum of sodium salicylate (Fig. 4) was obtained from the ratio of the two spectra shown in Fig. 3.

NOTE: The cut-off wavelength of L42 filter is 410 nm.  Therefore, in this example the correction procedure can only be applied to a wavelength range shorter than 410 nm.

 

Diffuse reflectance component (left), Diffuse reflectance spectrum of sodium salicylate (right)
Figure 3. Diffuse reflectance component (left), Figure 4. Diffuse reflectance spectrum of sodium salicylate (right)
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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