Fluorometer
Background
Significance
Fluorescence quantum yield determination using relavitive method
One significant use of the fluorometer (or fluorimeter) is the determination of the fluorescence quantum yield. This is done using a relative method based on a reference compound of known quantum yield. The unknown sample and the reference sample are measured at the same excitation wavelengths and measurement conditions. The wavelength-integrated flourescent intensity of both materials are then used in the calculation:
- <math>\phi = \phi_{reference} \frac {F_{sample} / A_{sample}} {F_{reference}/ A_{reference}} \left( \frac {n_{sample}} {n_{reference}} \right) ^2\,\!</math>
where
φ is the quantum yield
F= integrated fluorescence intensity
A= absorbance at excitation wavelength
n= refractive index
Optically dilute solution Intensity of excitation beam should be almost constant along excitation beam Fluorescence signal is proportional to intensity of excitation beam Intensity has to be the same for sample and reference Typically: A ≤ 0.02 over 1 cm pathlength From entrance face to center of cuvette: A = 0.01
Intensity has changed only by 2% Depending on absorption spectrometer used, measurement of A in this range may not be accurate enough How to proceed? Measure A on higher concentration solution Dilute solution by (accurately) known factor Perform fluorescence measurement on diluted solutions
Choice of excitation wavelength Excitation wavelength should be within the absorption band of the compounds Same excitation wavelength to be used for reference and sample compounds Emission spectrum collected on the long wavelength side of the excitation wavelength (to avoid strong scattered light from excitation beam) In this test, we are using: λexc = 350 nm Repeated measurements Prepare multiple dilutions Measure fluorescence emission spectrum of each solution Determine the slope of the line F/A for sample and reference Always a good idea to have multiple data points!
Deviations from linearity could indicate that emission was affected by reabsorption Reabsorption Absorption of the emitted light by the same solution before light exits cuvette More significant for compounds with small Stokes shifts Reabsorption can appear as a redshift (or decrease in fluorescence intensity on the short wavelength portion of the spectrum) Effect can be minimized by reducing concentration of solution Corrected fluorescence spectra Detectors and gratings do not have the same efficiency at all wavelengths Results need to be corrected by a factor that accounts for wavelength response of the instrument The contribution of the solvent (Raman scattering) and noise (dark counts) should also be subtracted Sample Calculation F/A values n Sample (#1): 1.289 x 1010 cps/mA 1.3288 (methanol) Reference: 1.316 x 1010 cps/mA 1.4266 (cyclohexane) φreference = 0.87)
- <math>\phi = \phi_{reference} \frac {F_{sample} / A_{sample}} {F_{reference}/ A_{reference}} \left( \frac {n_{sample}} {n_{reference}} \right) ^2\,\!</math>
- <math>\phi = 0.87 * \frac {1.289} {1.316} * \left( \frac {1.3288} {1.4266} \right) ^2\,\!</math>