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A Versatile Approach for Reliable Determination of Both High and Low Values of Luminescence Quantum Yields Krzysztof Nawara, Anup Rana, Pradeepta K. Panda, and Jacek Waluk Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02751 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 10, 2018

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Analytical Chemistry

A Versatile Approach for Reliable Determination of Both High and Low Values of Luminescence Quantum Yields Krzysztof Nawara1*, Anup Rana2, Pradeepta K. Panda2, Jacek Waluk1,3 1

Faculty of Mathematics and Science, Cardinal Stefan Wyszyński University, Dewajtis 5, 01-

815 Warsaw, Poland

2

School of Chemistry and Advance Centre of Research in High Energy Materials

(ACRHEM), University of Hyderabad, Hyderabad-500046, India 3

Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, Warsaw,

Poland

*

Correspondence to: [email protected]

Abstract The determination of luminescence quantum yields by means of relative methods requires setting identical experimental conditions for both, sample and reference compounds. This requirement has a critical impact on the applicability of these protocols, as it does not allow for the precise determination of low quantum yield values using well-characterized high quantum yield standards. We show that using the SAFE approach (Simultaneous Absorption and Fluorescence Emission measurement, Anal. Chem. 2017, 89 (17), 8650–8655), the sample and reference compounds can be effectively measured with different excitation slit spectral bandpass or integration times, separately optimized for each chromophore. This unique feature simplifies determination of luminescence quantum yields, allowing measurements of low quantum yield values using well-characterized high quantum yield standards.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Introduction Luminescence quantum yield is a crucial parameter characterizing both emissive and nonemissive properties of a chromophore in a given environment. Several reviews have been published describing protocols and methods used for luminescence quantum yield determination1–5. Recently, the number of reported quantum yield values greatly increased. Unfortunately, these values are often determined inaccurately. Remarkable discrepancies are revealed between the data for the same compound, but reported by different laboratories3,4. This creates an urgent need to develop methods and novel reference compounds that would minimize problems associated with the accuracy and reproducibility of the published quantum yield values. As pointed out by A. M. Brouwer, Chair of the IUPAC project: “Measurement of photoluminescence quantum yields”, it is especially desirable to provide standards for determination of low quantum yield values or methods which would deal with large differences in luminescence intensity between the sample and reference compounds4. In this work we propose an approach which accommodates these needs and allows for determination of low quantum yield values using well characterized high quantum yield reference standards. The basic feature of our method is the independence of the obtained quantum yield value on the spectral bandpass size of the excitation monochromator slit and the integration time of the measurement. The possibility of changing these experimental conditions is a unique characteristic of our approach, which is not recommended for other standard methods1,3. The protocol greatly facilitates determination of luminescence quantum yields, especially for very weakly emitting compounds. The recently developed SAFE method6 relies on simultaneous absorbance and fluorescence emission measurement for a given compound, which eliminates the number of problems previously described, such as: wavelength dependence (since both absorption factor and fluorescence emission are measured at exactly the same position of the excitation monochromator), the need for calculation of spectral integrals for absorption factors or trivial errors previously discussed6. The formula for quantum yield determination using the relative method is given by: ஺

ܻܳ௫ = ܻܳோ ೑ೌ೎೟೚ೝషೃ ௙

೔೙೟೐೒ೝೌ೗షೃ

మ ௙೔೙೟೐೒ೝೌ೗షೣ ௡ೣ మ ஺೑ೌ೎೟೚ೝషೣ ௡ೃ

(1)

where QYR is the quantum yield value of the reference compound, Afactor is the absorption factor, fintegral denotes the integrated emission intensity, n is the refractive index of the 2 ACS Paragon Plus Environment

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medium, and the subscripts R and x refer to the reference compound and the measured sample, respectively. The method requires determination of integrals of the emission spectra for both the reference and unknown samples, as well as the corresponding absorption factors, which are defined as: Afactor= 1- T-λ1 (2) where T is the sample transmittance at a given wavelength λ. The advantage of the SAFE method relies on the fact that both absorption factors and emission integrals are determined simultaneously for each sample with the same instrument. The ratio of

௙೔೙೟೐೒ೝೌ೗ ஺೑ೌ೎೟೚ೝ

is a direct, accurate measure of the luminescence quantum yield, since

both parameters are determined simultaneously. The intensity of an emission spectrum depends on the spectral bandpass of the slit of the excitation monochromator, as the fluorescence intensity is directly proportional to the intensity of incident light7. In this article we show that the emission spectrum (and its integral) corrected for the incident beam intensity (by the reference detector) is independent on the spectral bandpass of the excitation slit and scales linearly with integration time (expressed as the number of counts). Therefore, it is possible to vary incident light intensities for the compared samples even by the factor of 100. The determined Afactor is also excitation slitdependent, but since both the blank sample and the compound under investigation are measured for the same slit size, it can be easily determined, and its value includes the shape of the spectral profile of absorptivity. It has to be emphasized that for this approach it is critical that all the detectors (reference, transmittance and emission) must work in the linear response regime.

Materials and methods Fluorescent dyes were of the highest available purity: rhodamine 6G (Lambda Physics), rhodamine 101 (Lambda Physics). 2,3,6,7,12,13,16,17-octaethylporphycene (OEPc) and 2,3,6,7,12,13,16,17-octamethoxyporphycene (OMPc) were obtained and purified according to published procedures8,9. Spectral grade ethanol (Merck, Uvasol) was used for the measurements. All the dye solutions were prepared freshly before measurement. All the measurements were carried out in 10 x 10 mm path length Spectrosil quartz cuvettes (Starna) 3 ACS Paragon Plus Environment


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equipped with a magnetic stirrer. The sample concentrations were adjusted so that the absorbance did not exceed the value of 0.05 at the maximum of the lowest energy peak. The solutions were excited at the following wavelengths: 505 nm (rhodamine 6G), 545 nm (rhodamine 101), 575 nm (OEPc), 390 nm (OMPc). Fluorescence was recorded on an FS5 spectrofluorometer (Edinburgh Instruments), equipped with three standard detectors: (i) a single-photon counting detector for emission measurements (Peltier cooled photomultiplier R928P); (ii) a reference detector for exciting light intensity correction (UV-enhanced silicon photodiode), and (iii) a transmission detector for the determination of the absorption factor (UV-enhanced silicon photodiode). The instrument is equipped with two single grating Czerny-Turner monochromators (focal length 225 mm). The 150W CW ozone-free xenon lamp is used as an excitation light source. The temperature of the samples was set to 22 ⁰C and controlled by the Peltier module-controlled cuvette holder (–qpod model, Quantum North West), which was mounted directly into the FS5 spectrofluorometer, as previously described10. In order to achieve a stable response signal from all the detectors a warm-up time (with open shutters) of 2 h was maintained in all measurements. The more detailed procedure required to achieve a stable response signal is described in our previous article11. All steady-state fluorescence spectra were registered with the emission spectral bandpass set to 3 nm and a variable excitation spectral bandpass ranging from 1 to 11 nm. In the present study, polarizers were not introduced into the optical path of the instrument. The emission spectra were dark current-subtracted and corrected for (i) wavelength-dependent sensitivity of the detector; (ii) excitation light intensity (based on the reference signal and the silicon photodiode response curve). They were also scaled for the integration time of the measurement. The emission correction curve was checked by means of secondary emission standards12. The calibration lamp (HgAr from Ocean Optics) was used to control and calibrate the wavelength accuracy of monochromators. Absorption spectra were recorded on a Shimadzu UV 2700 double beam spectrophotometer All spectra were collected using 10x10 mm pathlength quartz cuvettes (Starna) with 1nm monochromator bandpass.

Determination of luminescence quantum yields


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The details of the procedure for the determination of luminescence quantum yield using simultaneous absorption and fluorescence emission (SAFE) approach have been published11. Here we are using the same protocol as described in the section Fluorescence quantum yield determination for the samples with a common absorption wavelength range11. In the literature often different values of luminescence quantum yield are reported even for the same reference compound. It is also the case for rhodamine 101 in ethanol solution, for which the following luminescence quantum yield values are reported: 0.90±0.0513, 0.915±0.02814, 0.9615,16, 1.00±0.0517. In this work for determination of luminescence quantum yields we have used the value for rhodamine 101 equal to 0.96 as the reference, as it allowed us to obtain the value of luminescence quantum yield for rhodamine 6G in ethanol solution equal to 0.93±0.03, which is in good agreement with literature: 0.8818 , 0.91±0.0414 , 0.9419,20 and 0.95±0.01521

The effect of integration time of the measurement and excitation monochromator beam slit size on the apparent value of luminescence quantum yield For the luminescence quantum yield measurement, the sample and blank are measured using identical parameters, including the same integration time and excitation slit bandpass size. The emission spectrum is corrected using the default intensity correction setting in Fluoracle software as well as for the sensitivity profile of the emission detector. The default correction of the intensity of the emission spectrum based on the reference channel signal is used to calculate the emission spectrum, which is independent of both, integration time of the measurement and the excitation slit bandpass size7. This correction also accounts for the different sensitivity of the reference detector as the function of wavelength, which is of primary importance when sample and reference are excited at different wavelengths. The standard deviation of the measurement was calculated from four measurements. All the spectral calculations and the error analysis were done using OriginPro2017 software.



Results and discussion Figure 1 demonstrates the effect of changing the excitation monochromator bandpass on the emission spectrum and the value of the absorption factor (the bandpass of the emission monochromator was constant and equal to 3nm).

A

B 120000

C

0.025

500000

300000

200000

0.024

Absorption factor

400000

Fluorescence intensity

100000

Fluorescence intensity

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80000

0.023

60000

0.022

40000

0.021

100000

20000

0

0.020

0 550 600 650 700

wavelength / nm

550 600 650 700

wavelength / nm

1

3

5

bandpass /nm

Figure 1: (a) Uncorrected emission spectra of rhodamine 6G in ethanol recorded for different excitation monochromator slit bandpasses: 1nm (black), 3nm (blue), and 5nm (red).(b) Corrected emission spectra of rhodamine 6G in ethanol recorded for different excitation monochromator slit bandpasses: 1nm (black), 3nm (blue), and 5nm (red).(c) Absorption factors calculated for the corresponding bandpasses of the excitation monochromator slit.

Figure 1a shows the uncorrected emission spectra of rhodamine 6G in ethanol measured for excitation slit bandpass sizes of 1, 3 and 5 nm, respectively. The change from 1 to 3 nm 6 ACS Paragon Plus Environment

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corresponds to the increase in the intensity of the emission signal by the factor of 10, whereas the intensity of the emission signal is enhanced by a factor of 27 upon variation of the spectral bandpass from 1 to 5 nm. Figure 1b is obtained using a default correction of light intensity (via reference channel) implemented in Fluoracle software. This correction is based on the signal which reaches the reference detector channel and therefore accounts for both timedependent factors (like the integration time of the measurement), as well as for the physical ones (like the size of the excitation bandpass). As a result, the intensity of the corrected emission spectrum becomes independent of excitation slit size and integration time. The increase of the integration time might, however, increase the precision of the determined values, as the signal to noise ratio increases with the integration time. Figure 1c show the values of the absorption factors obtained for the corresponding excitation slit values. The values of the absorption factors registered for different slit sizes do not have to be exactly the same, as they depend on the integral of the bandpass over absorption spectrum3, which is implicitly determined in the SAFE method, whereas for the other methods it has to be calculated according to the formula: భ మ భ ఒబ ିమ∆ఒ

‫ܣ‬௙௔௖௧௢௥ = ‫׬‬

ఒబ ା ∆ఒ

1 − 10ି஺ഊ ݀ߣ

(3)

where λ0 denotes the center of the excitation band, ∆λ is the excitation bandpass, and Aλ is the absorbance measured at λ. Figure 2 shows the values of luminescence quantum yield determined as the function of the slit bandpass size. These values were obtained using eq. (1) with rhodamine 101 in ethanol as the reference (QY=0.96)15,16, based on the values of

௙೔೙೟೐೒ೝೌ೗ ஺೑ೌ೎೟೚ೝ

for both compounds.



R101 R6G R6G OEPc OMPc

1.2E+00 1.1E+00 9.9E-01 9.0E-01 8.1E-01

quantum yield

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8.8E-03 8.6E-03 8.4E-03 8.2E-03 8.0E-03 1.7E-05 1.6E-05 1.5E-05 1.4E-05 1.3E-05 1

2

3

4

5

6

7

8

9

10

11

bandpass /nm

Figure 2: The calculated luminescence quantum yield values as the function of excitation monochromator slit bandpass for rhodamine 101 in ethanol (black), rhodamine 6G in ethanol (red), rhodamine 6G highly diluted solution in ethanol (blue), OEPc in ethanol (green), OMPc in ethanol (purple). The luminescence quantum yield values were determined using rhodamine 101 in ethanol as a standard (QY=0.96)15,16.

We recall here that, in order to correctly register the emission spectrum of any compound, several issues have to be considered, which may become extremely important when the sample and reference compounds strongly differ in quantum yields. First, there is a limit on the sample concentration. In order to avoid inner filter effects, the absorbance should not exceed the value of 0.05 at the maximum of the lowest energy absorption peak (secondary inner filter effect)3,7,22,23. On the other hand, too low absorbance at the excitation wavelength would produce high uncertainty in the absorption factor and, in consequence, in the luminescence quantum yield. Second, having established the concentration limits, the next 8 ACS Paragon Plus Environment

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factor to consider is the slit size of the excitation monochromator. For samples with high values of luminescence quantum yield the excitation slit cannot be too wide in order not to exceed the linear response of the emission detector channel. However, using too narrow slits for high luminescence quantum yield compounds can reduce precision in the absorption factor determination, as a too low signal may reach the transmittance detector. For samples with low values of the luminescence quantum yield too narrow excitation slits can cause a problem of no or very little signal at the emission detector. On the other hand, too wide slits for the compounds with low values of the luminescence quantum yields might produce the problem with saturation of the reference channel detector, leading to the failure in correcting the emission spectrum based on this channel, which is essential for the reliable luminescence quantum yield determination. Figure 2 shows that each compound, depending on its individual chemical and physical nature, should be measured using instrumental parameters which are the most suitable for it, allowing luminescence quantum yield measurement with highest possible precision. These limits are clearly seen for the rhodamine 6G solutions in ethanol. We have measured the standard rhodamine 6G ethanol solution with the absorbance value at the maximum below 0.05, with the excitation slits of 1, 3, and 5 nm, and the very diluted rhodamine 6G solution using excitation slits of 5, 8, and 10 nm. The high quantum yield value of rhodamine 6G does not allow (without dilution) to exceed an excitation slit size of 5 nm without reaching the nonlinear response regime of the emission detector channel. On the other hand, the slit size of 1 nm provides a reasonable emission signal, but the light intensity reaching the transmittance detector is fairly low, leading to higher uncertainty. The diluted solution of rhodamine 6G produces reasonable values at the emission channel, but the very low absorbance value at 505 nm leads to high uncertainty for all excitation slit size configurations for this sample. The terms “too wide”, “too narrow” are sample-dependent and can be determined only experimentally. Figure 2 also demonstrates a very important feature of the SAFE method. Apart from the issue of uncertainty, it clearly shows that changing the excitation slit size does not affect the mean value of the luminescence quantum yield (data for rhodamine 101, rhodamine 6G, and OEPc). Therefore, contrary to the situation encountered in standard relative measurements, it is possible using SAFE to determine the value of

௙೔೙೟೐೒ೝೌ೗ ஺೑ೌ೎೟೚ೝ

for the studied compound with the

maximum available precision (which is dependent on the excitation slit size). This value can 9 ACS Paragon Plus Environment


then be compared to the

௙೔೙೟೐೒ೝೌ೗ ஺೑ೌ೎೟೚ೝ

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value of the reference compound measured for different,

optimal experimental parameters and can be used for calculation of the luminescence quantum yields. In order to prove the effectiveness of this approach, we have employed it for the determination of quantum yield values of two very weakly emissive compounds: OEPc and OMPc. For ethanol solutions of OEPc the value of luminescence quantum yield was found to be equal to (8.4±0.1)⸱10-3 , which is in agreement with previous studies, although in different solvents24. We have also challenged our approach with the OMPc sample, which has the luminescence quantum yield value lower than

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