Skip to content

JASCO JASCO

  • News
  • Events
  • E-Store
  • My Account
  • Contact Us
  • Worldwide
Search
Click to view menu
MENUMENU
  • Products
    • Chromatography
      • HPLC
      • RHPLC
      • UHPLC
      • LC-MS
      • Preparative LC
      • Analytical SFC
      • Semi-Preparative SFC
      • Hybrid SFC
      • Fuel Analysis by SFC-FID
      • Preparative SFC
      • Supercritical Fluid Extraction
      • Chromatography Software
    • Molecular Spectroscopy
      • Circular Dichroism
      • High-Throughput CD
      • Vibrational CD
      • Circularly Polarized Luminescence (CPL)
      • Polarimeters
      • FTIR Spectrometers
      • FTIR Microscopy
      • FTIR Portable
      • Raman Microscopy
      • Palmtop Raman Spectrometer
      • Probe Raman
      • UV-Visible/NIR Spectrophotometers
      • UV-Visible/NIR Microscopy
      • Fluorescence Spectrophotometers
      • Film Thickness
      • Spectra Manager™ Suite
    • Refurbished
      • Refurbished HPLC Systems
      • HPLC Switching Valves
      • FTIR Accessories
  • Service
    • Service and Support Plans
    • Service Request Form
  • Applications
  • KnowledgeBase
  • Learning Center
    • Best Practice
      • Circular Dichroism Tips & Tricks for Biological Samples
      • CD Scale Calibration with ACS
      • Fluorescence Tips & Tricks
      • Raman Spectroscopy Tips & Tricks
    • Training Videos
      • ChromNAV
      • SF-NAV
      • Circular Dichroism
      • UV-Visible/NIR
      • Fluorescence
    • Training Seminars
      • Training Registration Form
    • Webinars
    • eBooks
    • Theory
      • Theory of Molecular Spectroscopy
      • Chromatography
  • About Us
    • President’s Message
    • Contact
    • History
    • Careers
  • News
  • Events
  • Worldwide
  • Shop
  • My Account
  • Contact Us

Home / Applications / High Sensitivity Coumarin Analysis

  • Industry

  • Technique

High Sensitivity Coumarin Analysis

By Heather Haffner

PDF IconDownload This Application

January 8, 2024

Introduction

To prevent the production of illegal light diesel oil, which contains kerosene or heavy oil, 1 ppm of coumarin is added to either the kerosene or a heavy oil as a discriminator. The analysis procedure for determining the discriminator and its mixing concentration is standardized by the National Petroleum Dealers Association and uses a spectrofluorometer to determine concentration.

Coumarin is hydrolyzed in alkaline solution and becomes cis-O-hydroxycinnamic acid. The cis-O-hydroxycinnamic acid is then isomerized by ultraviolet radiation and converted to trans-O- hydroxycinnamic acid. Since trans-O- hydroxycinnamic acid fluoresces, the product can be quantified using fluorescence spectroscopy.

Hydrolysis and photoisomerization of coumarin
Scheme 1. Hydrolysis and photoisomerization of coumarin.

 

While the current procedure is relatively simple, the purpose is to determine the coumarin concentration within more than a couple percent of the related oils. This application note introduces a high sensitivity system to improve the concentration detection limit to less than 1 percent and reduce the quantitation limit.

Experimental

1000 ppm of undiluted coumarin solution was made by dissolving 100 mg of coumarin in an aromatic solvent such as n-propyl benzene. The standard coumarin samples were prepared by diluting 100 µL of the undiluted coumarin solution using 1 ppm of n-dodecane and further diluting the solution to 0.1 ppm. For the alkaline solution reagent, 10 g of sodium hydroxide and 20 g of sodium nitrate were dissolved in 100 mL of Millipore water and kept in a polyethylene vessel. 40 mL of 1-butanol and 30 mL of ethanol were mixed for the alcohol solution. The standard samples were then mixed in test tubes, according to the specified ratios in Table 1. Following the procedure in Figure 2, the samples were shaken to hydrolyze the coumarin and then extracted in the alkaline solution. The samples were then kept stationary for 5 minutes to allow for separation of the solution layers. The photoisomerization reaction was induced by irradiating the sample using a spectrofluorometer and an excitation wavelength of 360 nm. The fluorescence intensity was detected at 500 nm and used to generate a calibration curve.

Table 1. Mixing ratios of the standard solution.

Additive ConcentrationCoumarin Solution (mL)n-dodecane (mL)Alkaline Solution (mL)Alcohol Dolution (ml)
0%04.234.8
1%0.064.1434.8
2%0.124.0834.8
4%0.243.9634.8
6%0.363.8434.8
8%0.483.7234.8
10%0.963.2434.8
Flow chart of the analysis procedure
Figure 1. Flow chart of the analysis procedure.

Measurement Conditions

FluorescenceTime Course
Excitation Wavelength360 nmExcitation Wavelength*360 nm
Scan Speed1000 nm/minEmission Wavelength500 nm
Excitation Bandwidth10 nmExcitation Bandwidth20 nm
Emission Bandwidth10 nmEmission Bandwidth10 nm
Data Interval1 nmData Interval2 sec
Response TimeFastResponse Time2 sec
SensitivityHighSensitivityHigh
Scan Speed500 nm/min

*The excitation was set at 20 nm to perform photoisomerization effectively for the Time course measurement. Excitation bandwidth was set at 10 nm for the Spectrum measurement in order to suppress reduction of fluorescence intensity due to photolysis.

Keywords

050-FP-0029B, FP-6300, Fluorescence, Coumarin, kerosene, diesel, O-hydroxycinnamic acid

Results

The time course and spectral measurement data of the standard samples with varying additive concentrations are shown in Figures 2 and 3. Both figures indicate an increase in fluorescence intensity with increasing additive concentration and Figure 2 shows that the photoisomerization reaction is finished in 150 seconds after exposing the samples to UV radiation.

Time Course measurement of the photoisomerization of the standard samples varying additive concentrations
Figure 2. Time Course measurement of the photoisomerization of the standard samples varying additive concentrations.
Fluorescence spectra after the photoisomerization reaction for varying additive concentrations
Figure 3. Fluorescence spectra after the photoisomerization reaction for varying additive concentrations.

A calibration curve plotting the fluorescence intensity at 500 nm as a function of the additive coumarin concentration is shown in Figure 4 and the corresponding values in Table 2. The correlation coefficient obtained for the calibration curve was 0.9993, indicating good linearity.

Table 2. The additive coumarin concentrations with their corresponding fluorescence intensities at 500 nm.

Additive ConcentrationFluorescence Intensity (at 500 nm)
0%6.7077
1%28.7548
2%57.3873
4%101.829
6%158.903
8%205.882
10%260.236
Calibration curve for the coumarin discriminator
Figure 4. Calibration curve for the coumarin discriminator.

The fluorescence measurements of 0% and 1% standard solution concentrations were repeated 5 times and are shown in Figure 5. The standard deviation for the fluorescence intensity was 0.4357 while the coumarin concentration was 0.0172.

Fluorescence spectra of 0% and 1% standard sample solutions. 5 measurements were taken for each concentration
Figure 5. Fluorescence spectra of 0% and 1% standard sample solutions. 5 measurements were taken for each concentration.

Conclusion

These results demonstrate that it is possible to use the high sensitivity spectrofluorometer system to analysis diesel oil discriminators with a 0.06% detection limit and 0.2% quantitation limit*.

*Detection limit was calculated by 3 sigma and quantitation limit was calculated by 10 sigma.

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.

Featured Products:

  • Simple and sensitive system which readily accommodates routine measurements and accessories, such as spectral scanning, quantitation, and temperature control.

    FP-8250 Spectrofluorometer

  • Sophisticated optical system offering the ultimate in sensitivity, spectral accuracy, and flexibility for the most challenging materials and biological samples.

    FP-8550 Spectrofluorometer

  • Uniquely optimized for NIR applications with extended wavelength measurement to 1010 nm.

    FP-8650 NIR Spectrofluorometer

About the Author

JASCO Application Note

High Sensitivity Coumarin Analysis

Introduction

To prevent the production of illegal light diesel oil, which contains kerosene or heavy oil, 1 ppm of coumarin is added to either the kerosene or a heavy oil as a discriminator. The analysis procedure for determining the discriminator and its mixing concentration is standardized by the National Petroleum Dealers Association and uses a spectrofluorometer to determine concentration.

Coumarin is hydrolyzed in alkaline solution and becomes cis-O-hydroxycinnamic acid. The cis-O-hydroxycinnamic acid is then isomerized by ultraviolet radiation and converted to trans-O- hydroxycinnamic acid. Since trans-O- hydroxycinnamic acid fluoresces, the product can be quantified using fluorescence spectroscopy.

Hydrolysis and photoisomerization of coumarin
Scheme 1. Hydrolysis and photoisomerization of coumarin.

 

While the current procedure is relatively simple, the purpose is to determine the coumarin concentration within more than a couple percent of the related oils. This application note introduces a high sensitivity system to improve the concentration detection limit to less than 1 percent and reduce the quantitation limit.

Experimental

1000 ppm of undiluted coumarin solution was made by dissolving 100 mg of coumarin in an aromatic solvent such as n-propyl benzene. The standard coumarin samples were prepared by diluting 100 µL of the undiluted coumarin solution using 1 ppm of n-dodecane and further diluting the solution to 0.1 ppm. For the alkaline solution reagent, 10 g of sodium hydroxide and 20 g of sodium nitrate were dissolved in 100 mL of Millipore water and kept in a polyethylene vessel. 40 mL of 1-butanol and 30 mL of ethanol were mixed for the alcohol solution. The standard samples were then mixed in test tubes, according to the specified ratios in Table 1. Following the procedure in Figure 2, the samples were shaken to hydrolyze the coumarin and then extracted in the alkaline solution. The samples were then kept stationary for 5 minutes to allow for separation of the solution layers. The photoisomerization reaction was induced by irradiating the sample using a spectrofluorometer and an excitation wavelength of 360 nm. The fluorescence intensity was detected at 500 nm and used to generate a calibration curve.

Table 1. Mixing ratios of the standard solution.

Additive ConcentrationCoumarin Solution (mL)n-dodecane (mL)Alkaline Solution (mL)Alcohol Dolution (ml)
0%04.234.8
1%0.064.1434.8
2%0.124.0834.8
4%0.243.9634.8
6%0.363.8434.8
8%0.483.7234.8
10%0.963.2434.8
Flow chart of the analysis procedure
Figure 1. Flow chart of the analysis procedure.

Measurement Conditions

FluorescenceTime Course
Excitation Wavelength360 nmExcitation Wavelength*360 nm
Scan Speed1000 nm/minEmission Wavelength500 nm
Excitation Bandwidth10 nmExcitation Bandwidth20 nm
Emission Bandwidth10 nmEmission Bandwidth10 nm
Data Interval1 nmData Interval2 sec
Response TimeFastResponse Time2 sec
SensitivityHighSensitivityHigh
Scan Speed500 nm/min

*The excitation was set at 20 nm to perform photoisomerization effectively for the Time course measurement. Excitation bandwidth was set at 10 nm for the Spectrum measurement in order to suppress reduction of fluorescence intensity due to photolysis.

Results

The time course and spectral measurement data of the standard samples with varying additive concentrations are shown in Figures 2 and 3. Both figures indicate an increase in fluorescence intensity with increasing additive concentration and Figure 2 shows that the photoisomerization reaction is finished in 150 seconds after exposing the samples to UV radiation.

Time Course measurement of the photoisomerization of the standard samples varying additive concentrations
Figure 2. Time Course measurement of the photoisomerization of the standard samples varying additive concentrations.
Fluorescence spectra after the photoisomerization reaction for varying additive concentrations
Figure 3. Fluorescence spectra after the photoisomerization reaction for varying additive concentrations.

A calibration curve plotting the fluorescence intensity at 500 nm as a function of the additive coumarin concentration is shown in Figure 4 and the corresponding values in Table 2. The correlation coefficient obtained for the calibration curve was 0.9993, indicating good linearity.

Table 2. The additive coumarin concentrations with their corresponding fluorescence intensities at 500 nm.

Additive ConcentrationFluorescence Intensity (at 500 nm)
0%6.7077
1%28.7548
2%57.3873
4%101.829
6%158.903
8%205.882
10%260.236
Calibration curve for the coumarin discriminator
Figure 4. Calibration curve for the coumarin discriminator.

The fluorescence measurements of 0% and 1% standard solution concentrations were repeated 5 times and are shown in Figure 5. The standard deviation for the fluorescence intensity was 0.4357 while the coumarin concentration was 0.0172.

Fluorescence spectra of 0% and 1% standard sample solutions. 5 measurements were taken for each concentration
Figure 5. Fluorescence spectra of 0% and 1% standard sample solutions. 5 measurements were taken for each concentration.

Conclusion

These results demonstrate that it is possible to use the high sensitivity spectrofluorometer system to analysis diesel oil discriminators with a 0.06% detection limit and 0.2% quantitation limit*.

*Detection limit was calculated by 3 sigma and quantitation limit was calculated by 10 sigma.

Keywords

050-FP-0029B, FP-6300, Fluorescence, Coumarin, kerosene, diesel, O-hydroxycinnamic acid

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.
28600 Mary’s Court, Easton, MD 21601 USA • (800) 333-5272 • Fax: (410) 822-7526 • jascoinc.com/applications

Close

Designed in Tokyo. TRUSTED globally.

View our support plans

Connect with JASCO

  • Facebook
  • Twitter
  • LinkedIn
  • JASCO Sales
  • 800-333-5272

Receive the latest promotions and special offers

  • This field is for validation purposes and should be left unchanged.
  • Careers
  • Press Kit
  • JASCO Privacy Policy
  • Sitemap
  • Environmental Policy

© , JASCO. All Rights Reserved.