Skip to content
Skip to content
  • News
  • Events
  • eBooks
  • Blog
  • Careers
  • Contact
  • News
  • Events
  • eBooks
  • Blog
  • Careers
  • Contact
KNOWLEDGEBASE
  • About Us
  • Products

    Fluorescence Spectrometers

    • FLS1000 Photoluminescence Spectrometer
    • FS5 Spectrofluorometer
    • LifeSpec II Lifetime Spectrometer
    • Mini-tau Lifetime Spectrometer

    Raman Microscopes

    • RM5 Raman Microscope
    • RMS1000 Multimodal Confocal Microscope

    Transient Absorption

    • LP980 Transient Absorption Spectrometer

    FTIR Spectrometers

    • IR5 FTIR Spectrometer

    Lasers and LEDs

    • Pulsed Lasers
    • Gas Lasers
    • Customisation Options
    View All Products
  • Techniques
  • Applications
KNOWLEDGEBASE
Edit Content
  • About Us
  • Products
  • Techniques
  • Applications
  • Knowledgebase
  • eBooks
  • News
  • Events
  • Blog
  • Careers
  • Contact Us

RESOURCES

Relative Quantum Yield of 2-Aminopyridine

  • April 14, 2022
Edit Content

Quantum yield is a fundamental photophysical parameter that describes a sample’s fluorescence efficiency and is defined as the ratio of the number of photons emitted to the number of photons absorbed by a sample. Accurate and reliable quantum yield measurements are important for a broad range of applications including displays, solar cells, bioimaging and drug development.

There are two optical methods for measuring the quantum yield: the absolute method and the relative method. In the absolute method, the quantum yield is measured directly using an integrating sphere, while in the relative method the fluorescence intensity of the unknown sample is compared with the fluorescence intensity of a standard sample to calculate the quantum yield of the unknown.

In this application note, an Edinburgh Instruments FS5 Spectrofluorometer (Figure 1) is used to measure the quantum yield of 2-Aminopyridine (2AMP) via the relative method. 2AMP in sulfuric acid (H2SO4) has been previously used as a quantum yield reference standard in the UV-visible range. The quantum yield of 2AMP was measured to be 60% in 19681 and 66% in 1983.2 These literature quantum yield reference values are now decades old, and here we present a reinvestigation and revaluation of the quantum yield of 2AMP in 1M H2SO4 using quinine bisulphate (QBS) in 1M H2SO4 as the reference standard with a modern spectrofluorometer.

Edinburgh Instruments FS5 Spectrofluorometer

Figure 1: Edinburgh Instruments FS5 Spectrofluorometer.

 

Methodology

The relative quantum yield of 2 Aminopyridine can be calculated through the following formula,3

Equation to calculate relative quantum yield of 2 Aminopyridine

Eq.1

where the subscripts S and R denote sample of interest (2AMP) and reference standard (QBS), respectively. Φ is the quantum yield, I is the integrated fluorescence intensity, and A is the absorbance at the excitation wavelength. n is the refractive index of the solvents used for sample and reference solutions at the mean emission wavelength. In this application note, the same solvent (1 M H2SO4) was used for both 2AMP and QBS and this term cancels.

To increase the accuracy and precision of the calculated quantum yield value, it is best practice to prepare and measure several solutions of the sample and reference with different concentrations. By plotting I versus 1-10-A for 2AMP and QBS, the gradients (GradS and GradR) can be used to calculate the quantum yield (Eq.2). This approach prevents potential systematic errors, such as dye aggregation, which would appear as a deviation from the straight line at higher concentrations.

2-Aminopyridine Equation

Eq.2

Five solutions of 2AMP in 1M H2SO4 and five solutions of QBS in 1M H2SO4 at different concentrations were prepared. Absorption and fluorescence spectra were measured using an FS5 Spectrofluorometer equipped with a 150 W Xenon lamp, a PMT-980 detector and a SC‑05 cuvette holder.

 

Absorption & Emission Spectra of 2-Aminopyridine & QBS

Firstly, the absorbance values of the five 2AMP and QBS solutions were determined by measuring the absorption spectra using the FS5’s built-in transmission detector. The absorbance values of the solutions were kept below 0.1 at the excitation wavelength (310 nm) to minimise the probability of inner filter effects and ranged between 0.008 and 0.098. The normalised absorption spectra of 2AMP and QBS are shown in Figure 2a.

Normalised absorbance and emission spectra of 2AMP and QBS and emission spectra of 2AMP at different concentrations

Figure 2: (a) Normalised absorbance spectra of 2AMP (green) and QBS (violet). (b) Normalised fluorescence spectra of 2AMP (green) and QBS (violet). (c) Fluorescence spectra of 2AMP of different concentrations. The C1 solution is the least concentrated (absorbance at 310 nm=0.01), and C5 is the most concentrated (absorbance at 310 nm=0.098). All spectra were acquired on an Edinburgh Instruments FS5 Spectrofluorometer.

Next, the fluorescence spectra of five 2AMP and QBS solutions were acquired. The intensity of the fluorescence detected by a spectrofluorometer is dependent on the excitation wavelength, excitation and emission bandwidths, and integration time. By keeping these parameters identical the integrated fluorescence intensities, IS and IR, of 2AMP and QBS can be meaningfully compared. The experimental parameters were λex=310 nm with excitation and emission bandwidths set at 3 nm and 0.5 nm, respectively, 1 nm step size, and 0.5 s integration time. Figure 2b shows the normalised fluorescence spectra of 2AMP and QBS.

 

Using Fluoracle Trend Analysis to Determine Quantum Yield

The fluorescence spectra for each concentration of 2AMP were combined into a single plot in Fluoracle (Figure 2c). The gradient GradS in Eq.2 can be calculated from the 2AMP spectra in Figure 2c using the Fluoracle Trend Analysis feature which is shown in Figure 3.

To calculate GradS the Calibration parameter was set to Area (orange frame) and (optionally) the Variable name was set as 1‑10‑A (green frame). The areas (integrated fluorescence intensity) are calculated by pressing Apply. The absorbance term (1-10-A) values for each concentration of 2AMP that were obtained from the absorption spectra were then input (light blue frame).

Trend Analysis feature in Fluoracle | Application Note: Relative Quantum Yield of 2-Aminopyridine

Figure 3: Figure 2c’s Trend Analysis in Fluoracle.

The Calibration type was set to Linear and the Force curve through zero box ticked (dark blue frame). The integrated fluorescence intensity versus absorbance term is plotted on the lower right side of the screen, along with the linear fit. The gradient of the curve (GradS) is given as K1 (red frame). The same process was then repeated for the five QBS spectra to calculate the gradient GradR. Both curves and their calculated gradients are shown in Figure 4.

Integrated fluorescence intensity versus absorbance term of 2AMP & QBS | Application Note: Relative Quantum Yield of 2-Aminopyridine

Figure 4: Integrated fluorescence intensity versus absorbance term of 2AMP and QBS.

The literature value of the quantum yield of QBS in H2SO4 is ΦR=56.1%.4 The quantum yield of 2AMP in H2SO4 was then calculated using Eq.2 to be 64.3%, a value which closely agrees with previous values reported of 60% and 66%.1,2

 

Conclusion

An Edinburgh Instruments FS5 Spectrofluorometer was used for the determination of the quantum yield of 2-Aminopyridine in 1M H­2SO4 via the relative method. The data analysis was made simple by the Trend Analysis feature of FS5 Fluoracle software. The quantum yield of 2-Aminopyridine, using Quinine Bisulphate as the reference standard, was calculated to be 64.3%. This value closely agrees with previous reports in the literature and demonstrates that the FS5 can undertake accurate and reliable relative quantum yield measurements.

 

References

  1. R. Rusakowicz, A. C. Testa, 2-Aminopyridine as a standard for low-wavelength spectrofluorimetry. J. Phys. Chem. 72, 2680–2681 (1968).
  2. S. R. Meech, D. Phillips, Photophysics of some common fluorescence standards. J. Photochem. 23, 193–217 (1983).
  3. K. L. Wong, J. C. Bünzli, P. A. Tanner, Quantum yield and brightness. J. Lumin. 224, 117256 (2020).
  4. B. Gelernt, A. Findeisen, A. Stein, J. A. Poole, Absolute measurement of the quantum yield of quinine bisulphate. J. Chem. Soc. Faraday Trans. 2 Mol. Chem. Phys. 70, 939–940 (1974).

RELATED PRODUCTS

DS5

Dual Beam UV-Vis Spectrophotometer

VIEW

FLS1000

Photoluminescence Spectrometer

VIEW

FS5

Spectrofluorometer

VIEW

Contact our expert team today to find out more about how our products can improve your research

Contact Us
PrevPrevious
NextNext
Previous Sb to Mn Energy Transfer Revealed using Time-Resolved Emission Spectroscopy Next A Short Introduction to Fluorescence Measurements & Instrumentation

RESOURCES

Tags:
  • Application Notes
  • Photophysics
  • FLS1000
  • FS5
  • Photoluminescence
Download PDF
Share:

Keep up to date with the latest from Edinburgh Instruments

Join our mailing list and keep up with our latest videos, app notes and more!

LOCATION:
  • Edinburgh Instruments Ltd.
    2 Bain Square, Kirkton Campus, Livingston, EH54 7DQ.
  • sales@edinst.com
  • +44 1506 425 300
ABOUT:
  • About Us
  • Techniques
  • Applications
  • Knowledgebase
  • About Us
  • Techniques
  • Applications
  • Knowledgebase
PRODUCTS:
  • Fluorescence Spectrometers
  • Raman Microscopes
  • UV-Vis Spectrophotometers
  • Transient Absorption
  • FTIR Spectrometers
  • Lasers and LEDs
  • Customisation Options
  • Software
  • Upgrades
  • All Products
  • Fluorescence Spectrometers
  • Raman Microscopes
  • UV-Vis Spectrophotometers
  • Transient Absorption
  • FTIR Spectrometers
  • Lasers and LEDs
  • Customisation Options
  • Software
  • Upgrades
  • All Products
LEGALS:
  • News
  • Events
  • Blog
  • Careers
  • Contact Us
  • Terms and Conditions
  • Privacy Policy
  • News
  • Events
  • Blog
  • Careers
  • Contact Us
  • Terms and Conditions
  • Privacy Policy
SOCIALS:
Youtube Linkedin X-twitter Facebook
©2024 Edinburgh Instruments. Registered in England and Wales No: 962331. VAT No: GB 271 7379 37
Manage Consent
To provide the best experiences, we use technologies like cookies to store and/or access device information. Consenting to these technologies will allow us to process data such as browsing behaviour or unique IDs on this site. Not consenting or withdrawing consent, may adversely affect certain features and functions.
Functional Always active
The technical storage or access is strictly necessary for the legitimate purpose of enabling the use of a specific service explicitly requested by the subscriber or user, or for the sole purpose of carrying out the transmission of a communication over an electronic communications network.
Preferences
The technical storage or access is necessary for the legitimate purpose of storing preferences that are not requested by the subscriber or user.
Statistics
The technical storage or access that is used exclusively for statistical purposes. The technical storage or access that is used exclusively for anonymous statistical purposes. Without a subpoena, voluntary compliance on the part of your Internet Service Provider, or additional records from a third party, information stored or retrieved for this purpose alone cannot usually be used to identify you.
Marketing
The technical storage or access is required to create user profiles to send advertising, or to track the user on a website or across several websites for similar marketing purposes.
Manage options Manage services Manage {vendor_count} vendors Read more about these purposes
View preferences
{title} {title} {title}