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 Fluorescence-Detected Circular Dichroism (FDCD) Measurement

  • Industry

  • Technique

High-Sensitivity Fluorescence-Detected Circular Dichroism (FDCD) Measurement

By Heather Haffner

PDF IconDownload This Application

January 5, 2024

Introduction

Fluorescence-detected circular dichroism (FDCD) can be used to measure the difference in fluorescence intensities when an optically active sample has been excited with circularly polarized light. FDCD offers superior selectivity in that it allows the user to measure the circular dichroism of a specific fluorescent chromophore in a group of nonfluorescent, chiral molecules. During an FDCD measurement, the molar circular dichroism can be calculated from the FDCD results and the following equation:

Δε = εl- εr= (3.032 ∙ 10-5) ∙ S ∙ (1 – 10-A) / c ∙ l ∙ 10-A) [1]

where εl – εr is the difference between left and right circular dichroism, S is the measured FDCD value, A is the absorbance of the sample, c is the concentration and l is the cell’s path length.

The JASCO FDCD-465 is a highly sensitive FDCD measurement accessory which combines both a cylindrical cell and an elliptical cylinder mirror (Figure 1). This design allows for the collection of all radiated fluorescence light which is emitted in a circumferential direction from the cell. The result not only improves the light collection efficiency, but also the ability to eliminate the artifacts effects caused by fluorescence anisotropy. [2] [3]

Solvents with high viscosities can also contribute to anisotropy effects, inducing artifacts. To further minimize these effects, the FDCD-465 includes a balancing mask. If the fluorescence light in the F-y direction does not reach the detector due to a blockage from the cell itself, the fluorescence intensity of F+x + F-x can become slightly greater than F+y + F-y, which could cause an artifact. By using the balancing mask, the intensities of these fluorescence signals can be balanced to eliminate the artifacts, even when using solvents with high viscosity.

In this application note, the J-1500 Circular Dichroism spectrometer and FCDC-465 accessory were used to measure ammonium d-10-camphorsulfonic acid in water.

J-1500 Circular Dichroism Spectrometer
J-1500 CD Spectrometer
Exterior and interior view of the FDCD-465 and its components
Figure 1. Exterior and interior view of the FDCD-465 and its components

Experimental

Measurement conditions
FDCD
CD
 Spectral bandwidth 4 nm Spectral bandwidth 2 nm
 Accumulations 16 times Accumulations 8 times

Keywords

260-CD-0006, J-1500, J-820, Circular Dichroism, CD, fluorescence-detected circular dichroism, FDCD-465, fluorescence, biochemistry

Results

Since the FDCD is an excitation spectrum, the excitation and emission wavelengths need to be obtained using a fluorescence spectrometer prior to FDCD measurements. An excitation wavelength of 285 nm was obtained from the fluorescence experiments along with a fluorescence emission maxima at 427 nm. These wavelengths were then used to measure the FDCD of 0.0024 M ammonium d-10-camphorsulfonic acid (d-10-ACS), which is shown in Figure 2.

Fluorescence Detected Circular Dichroism (top), Circular Dichroism (middle), and absorption (bottom) spectra of 0.0024 M d-10-ACS in water was obtained using a L38 excitation light cut-off filter and a 1 cm pathlength cell.
Figure 2. FDCD (top), CD (middle), and absorption (bottom) spectra of 0.0024 M d-10-ACS in water was obtained using a L38 excitation light cut-off filter and a 1 cm pathlength cell.

Based on the obtained FDCDC, CD, and absorption spectra, the CD spectrum was converted from FDCD using equation 1. A comparison of a measured CD spectrum and CD spectrum calculated from FDCD measurements is shown in Figure 2 and depicts that the spectra are in good agreement with each other.

 Comparison of the measured Circular Dichroism spectrum (lilac) and Circular Dichrosim spectrum calculated from FDCD data (magenta).
Figure 3. Comparison of the measured CD spectrum (lilac) and CD spectrum calculated from FDCD data (magenta).

Figure 4 shows the FDCD and CD spectra of (1S,2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) and acetonitrile solution at varying concentrations, respectively. For the FDCD data (left), the difference in the fluorescence intensity excited by circularly polarized light is normalized against the intensity of the total fluorescence. Therefore, the FDCD signal (mdeg) shown in Figure 4 displays a constant value regardless of sample concentration.

In the CD measurements, a CD signal could only be observed with a concentration of 2 · 10-7 M. The concentrations at 2 · 10-8 M and 2 · 10-9 M did not show usable CD signals. However, using the FDCD-465 accessory for spectral measurements, a signal was clearly observed with a concentration two orders of magnitude lower than 2 · 10-7M.

FDCD of (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) (left) and CD of acetonitrile (right) spectra at varying low concentrations. We kindly thank Tatsuo Nehira (Hiroshima University) and Katsunori Tanaka (Osaka University) for providing us with information regarding (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate).
Figure 4. FDCD of (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) (left) and CD of acetonitrile (right) spectra at varying low concentrations. We kindly thank Tatsuo Nehira (Hiroshima University) and Katsunori Tanaka (Osaka University) for providing us with information regarding (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate).

Conclusion

This application note demonstrates that if a sample is amendable to FDCD measurement, much higher selectivity and sensitivity can be achieved compared to standard CD measurements.

References

  1. Turner, D.H., Tinoco Jr, I., and M. Maestre, (1974) JACS, 96, 4340-4342.
  2. Nehira, T., tanaka, K., Takakuwa, T., Ohshima, C., Masago, H., Pescitelli, G., Wada, A., and N. Berova, (2005) Applied Spectroscopy, 59, 121-125.
  3. Tanaka, K., Pescitelli, G., Nakanishi, K., and N. Berova, (2005) Monatshefte für Chemie/Chemical Monthly, 136, 367-395.
  4. T. Nehira, (2005) Monatshefte für Chemie/Chemical Monthly, 136, 447-487.
  5. Dong, J.G., Wada, A., Takakuwa, T., Nakanishi, K., and N. Berova, (1997) JACS, 119, 12024-12025.
  6. Muto, K., Mochizuki, H., Yoshida, R., Ishii, T., and T. Handa, (1986) JACS, 108, 6416-6417.
  7. 7. Nehira, T., Parish, C. A., Jochusch, S., Turro, N. J., Nakanishi, K., and N. Berova, (1999) JACS, 121, 8681-8691.
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:

  • The basic, compact model for research, QA/QC, and teaching applications.

    J-1100

  • Highest performance with a wide range of accessories for maximum flexibility to meet complex research demands.

    J-1500

  • UV/Visible/NIR measurements up to 2,500 nm for MCD and specialized applications.

    J-1700

About the Author

JASCO Application Note

High-Sensitivity Fluorescence-Detected Circular Dichroism (FDCD) Measurement

Introduction

Fluorescence-detected circular dichroism (FDCD) can be used to measure the difference in fluorescence intensities when an optically active sample has been excited with circularly polarized light. FDCD offers superior selectivity in that it allows the user to measure the circular dichroism of a specific fluorescent chromophore in a group of nonfluorescent, chiral molecules. During an FDCD measurement, the molar circular dichroism can be calculated from the FDCD results and the following equation:

Δε = εl- εr= (3.032 ∙ 10-5) ∙ S ∙ (1 – 10-A) / c ∙ l ∙ 10-A) [1]

where εl – εr is the difference between left and right circular dichroism, S is the measured FDCD value, A is the absorbance of the sample, c is the concentration and l is the cell’s path length.

The JASCO FDCD-465 is a highly sensitive FDCD measurement accessory which combines both a cylindrical cell and an elliptical cylinder mirror (Figure 1). This design allows for the collection of all radiated fluorescence light which is emitted in a circumferential direction from the cell. The result not only improves the light collection efficiency, but also the ability to eliminate the artifacts effects caused by fluorescence anisotropy. [2] [3]

Solvents with high viscosities can also contribute to anisotropy effects, inducing artifacts. To further minimize these effects, the FDCD-465 includes a balancing mask. If the fluorescence light in the F-y direction does not reach the detector due to a blockage from the cell itself, the fluorescence intensity of F+x + F-x can become slightly greater than F+y + F-y, which could cause an artifact. By using the balancing mask, the intensities of these fluorescence signals can be balanced to eliminate the artifacts, even when using solvents with high viscosity.

In this application note, the J-1500 Circular Dichroism spectrometer and FCDC-465 accessory were used to measure ammonium d-10-camphorsulfonic acid in water.

J-1500 Circular Dichroism Spectrometer
J-1500 CD Spectrometer
Exterior and interior view of the FDCD-465 and its components
Figure 1. Exterior and interior view of the FDCD-465 and its components

Experimental

Measurement conditions
FDCD
CD
 Spectral bandwidth 4 nm Spectral bandwidth 2 nm
 Accumulations 16 times Accumulations 8 times

Results

Since the FDCD is an excitation spectrum, the excitation and emission wavelengths need to be obtained using a fluorescence spectrometer prior to FDCD measurements. An excitation wavelength of 285 nm was obtained from the fluorescence experiments along with a fluorescence emission maxima at 427 nm. These wavelengths were then used to measure the FDCD of 0.0024 M ammonium d-10-camphorsulfonic acid (d-10-ACS), which is shown in Figure 2.

Fluorescence Detected Circular Dichroism (top), Circular Dichroism (middle), and absorption (bottom) spectra of 0.0024 M d-10-ACS in water was obtained using a L38 excitation light cut-off filter and a 1 cm pathlength cell.
Figure 2. FDCD (top), CD (middle), and absorption (bottom) spectra of 0.0024 M d-10-ACS in water was obtained using a L38 excitation light cut-off filter and a 1 cm pathlength cell.

Based on the obtained FDCDC, CD, and absorption spectra, the CD spectrum was converted from FDCD using equation 1. A comparison of a measured CD spectrum and CD spectrum calculated from FDCD measurements is shown in Figure 2 and depicts that the spectra are in good agreement with each other.

 Comparison of the measured Circular Dichroism spectrum (lilac) and Circular Dichrosim spectrum calculated from FDCD data (magenta).
Figure 3. Comparison of the measured CD spectrum (lilac) and CD spectrum calculated from FDCD data (magenta).

Figure 4 shows the FDCD and CD spectra of (1S,2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) and acetonitrile solution at varying concentrations, respectively. For the FDCD data (left), the difference in the fluorescence intensity excited by circularly polarized light is normalized against the intensity of the total fluorescence. Therefore, the FDCD signal (mdeg) shown in Figure 4 displays a constant value regardless of sample concentration.

In the CD measurements, a CD signal could only be observed with a concentration of 2 · 10-7 M. The concentrations at 2 · 10-8 M and 2 · 10-9 M did not show usable CD signals. However, using the FDCD-465 accessory for spectral measurements, a signal was clearly observed with a concentration two orders of magnitude lower than 2 · 10-7M.

FDCD of (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) (left) and CD of acetonitrile (right) spectra at varying low concentrations. We kindly thank Tatsuo Nehira (Hiroshima University) and Katsunori Tanaka (Osaka University) for providing us with information regarding (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate).
Figure 4. FDCD of (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) (left) and CD of acetonitrile (right) spectra at varying low concentrations. We kindly thank Tatsuo Nehira (Hiroshima University) and Katsunori Tanaka (Osaka University) for providing us with information regarding (1S, 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate).

Conclusion

This application note demonstrates that if a sample is amendable to FDCD measurement, much higher selectivity and sensitivity can be achieved compared to standard CD measurements.

Keywords

260-CD-0006, J-1500, J-820, Circular Dichroism, CD, fluorescence-detected circular dichroism, FDCD-465, fluorescence, biochemistry

References

  1. Turner, D.H., Tinoco Jr, I., and M. Maestre, (1974) JACS, 96, 4340-4342.
  2. Nehira, T., tanaka, K., Takakuwa, T., Ohshima, C., Masago, H., Pescitelli, G., Wada, A., and N. Berova, (2005) Applied Spectroscopy, 59, 121-125.
  3. Tanaka, K., Pescitelli, G., Nakanishi, K., and N. Berova, (2005) Monatshefte für Chemie/Chemical Monthly, 136, 367-395.
  4. T. Nehira, (2005) Monatshefte für Chemie/Chemical Monthly, 136, 447-487.
  5. Dong, J.G., Wada, A., Takakuwa, T., Nakanishi, K., and N. Berova, (1997) JACS, 119, 12024-12025.
  6. Muto, K., Mochizuki, H., Yoshida, R., Ishii, T., and T. Handa, (1986) JACS, 108, 6416-6417.
  7. 7. Nehira, T., Parish, C. A., Jochusch, S., Turro, N. J., Nakanishi, K., and N. Berova, (1999) JACS, 121, 8681-8691.
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.