Fluorescent Quantum Yields for Dummies
By Juan C. Castro
Burgess Group
April 17th, 2006
The determination of quantum yield requires previous preparation.
A few points that must be followed to obtain good quantum yields are;
•
If this is the first time you are attempting to obtain a quantum yield measurement,
it is recommended to use two known standards, and cross-reference their quantum
yields to get use to the procedure prior to determining the value for an unknown.
•
It is very important to use a well maintained and calibrated UV/Vis and
fluorometer, with high sensitivity and low signal-to-noise ratios.
•
An adequate fluorescent yield standard must be chosen to ensure maximum
overlap of the absorption between the unknown sample and reference standard.
•
Once the spectrofluorometer parameters have been adjusted and set, they should
not be changed during the entire experiment.
•
To minimize errors for measured absorption values, the same cuvette should be
used for both absorption and emission determinations for the unknown and the
standard, that is, only one cuvette should be used throughout.
•
To avoid inner filter effects, the absorbance values obtained must be bellow 0.1
at the excitation wavelength, preferably, within 0.01 and 0.05, although lower
values than 0.01 could be used if a good absorption spectra is obtained, and it is
high enough for the spectrofluorometer to detect its emission.
•
It is advisable to calculate quantum yields utilizing the same solvent for both the
unknown and the standard. It is possible to correct for the different refractive
indexes if different solvent are used, as seen in equation 1, but this introduces a
new variable which may cause the quantum yield values obtained to be less
accurate.
•
Be also aware, that the obtained emission spectra MUST be corrected to obtain
good measurements. This is usually accomplished by applying a correction file to
the obtained spectra, either before or after the fluorescent measurement. When in
doubt, ask a person knowledgeable about the instrument.
STEP by STEP determination of quantum yields
1) A good standard must first be chosen, a list of available standards is given in
Figure 1. These standards have a quantum yield independent of excitation
wavelength, and therefore, a suitable excitation wavelength could be
accommodated to excite both the unknown and standard.
Figure 1. UV/Vis of selected QY standards
2) When selecting a good excitation wavelength to be used, it is better to obtain a
crude absorption spectra of the unknown and the standard, and overlap them. A
good wavelength must have a relatively high absorbance value for each solution.
We have found that choosing a standard with a good absorbance close to the λmax
of the unknown provides excellent results.(figure 2)
Figure 2. Sample determination of good excitation wavelength
3) To prepare the solutions, it is imperative to use clean glassware, and to be
cautious about cross contaminating the samples with the other solution. This
method for determination of quantum yields is independent on the concentration
of the samples, which do not have to be the same, but nonetheless, to conserve the
supply of standards, no more that 2 mg of each compound must be used.
a. Preparation of solutions
i. Weigh about 2 mg of the compound and place inside a 3 ml glass
vial, preferably amber color to avoid photobleaching, and cap
tightly.
ii. Add 2 ml of high quality solvent. Mix in vortex to dissolve any
residue.
iii. Prepare at least four glass test tubes, and add 2 ml of the same
solvent used in step ii.
iv. To the first test tube, add 200 µl of the dye solution prepared in
step ii. mix well.
v. To the second test tube, add 100 µl of the solution prepared in step
iv. mix well
b. After step v. in the previous section, an almost colorless solution is
obtained. This may be the right concentration to be used, but it must first
be checked for a good absorbance value at the excitation wavelength
4) Carefully run a blank using the same cuvette that is to be used with the
compounds. Make sure to use exactly the same solvent.
5) After thoroughly cleaning the cuvette, proceed to obtain an absorption spectra of
the unknown.
6) Measure the aborbance at the excitation wavelength to be used for both standard
and unknown and subtract from this the 'baseline" from either point of the same
spectra as shown in figure 3, in this case, the right "height" of the specific
wavelength is obtained, avoiding errors by a drifting instrument baseline.
Figure 3. Determination of actual absorbance reading at excitation wavelength
7) If the measured absorbance is equal or less than 0.01, proceed to obtain
fluorescence, as long as a clearly defined curve is observed, if not, either add or
dilute solution until a good value is achieved.
8) By using the same cuvette, proceed to obtain an emission spectra. If using an ISS
PC1 instrument, make sure to mark the option to correct the file "without
polarimetet and 0.5 slits", and make sure a 0.5 slit is present at the PMT
(photomultiplier tube) end. Obtain a suitable wavelength range which would
completely contain the spectra for both the unknown sample and standard, and
keep it the same for both.
9) Obtain the integrated emission spectra for each compound within the same limits
for both (ie. 500 - 650 nm).
10) Repeat the same procedure with the known fluorescence standard.
11) Plot the obtained values in the following equation.
Equation 1
φx = φst (Ix/Ist)(Ast/Ax)(nx2/nst2)
Where φ st is the reported quantum yield of the standard, I is the integrated emission
spectra, A is the absorbance at the excitation wavelength and n is the refractive index
of the solvent used (n=1 if same solvent). X subscript denotes unknown, and st
denotes standard.
12) You may know compare your obtained quantum yields if you used two standards,
and make sure you are within 10 % of the reported value, this is good, but could
be better. With time and practice, a better value could be obtained. Any value
above 1.0 is not allowed, although in practice measurements a value less than or
equal to 1.15 is possible. Once good values are obtained when cross-referencing
standards, measurements of unknown quantum yields may be performed.
THE END