Russian Physics Journal, Vol. 56, No. 12, April, 2014 (Russian Original No. 12, December, 2013)
pH-DEPENDENCE OF THE ABSORPTION AND FLUORESCENT PROPERTIES OF FLUORONE DYES IN AQUEOUS SOLUTIONS E. A. Slyusareva and M. A. Gerasimova
UDC 535.37; 535.34; 543.428
A series of fluorone dyes (fluorescein, eosin Y, erythrosin B) in aqueous solution is investigated by the absorption, fluorescence spectroscopic and time-resolved methods. Based on an analysis of the absorption spectrum amplitude, the fluorescence quantum yield, and the fluorescence lifetime vs. pH, the dissociation constants of the dyes in the ground and excited states are calculated. Quantitative and qualitative differences in the character of ionic equilibrium between fluorescein and its halogenated derivatives – eosin Y and erythrosin B – are revealed. The polarized fluorescence method has shown that the hydrodynamic diameter changes in a series of fluorone dyes due to both the increase of the bond length upon halogenation and the influence of solvation shell upon the change of the dye ionic species. Keywords: fluorescein, eosin Y, erythrosin B, ionic species, absorption spectra, fluorescence spectra, fluorescence lifetime, polarization degree, dissociation constant.
INTRODUCTION Fluorone dyes represent a homologous series of compounds based on fluorescein with gradual substitution of hydrogen atoms by halogen atoms (Fig. 1). The possibility of successful application of fluorone dyes for biochemical probes [1–4] is caused by both their luminescent properties and presence of step dissociation with manifestation of spectral variety of different ionic states. An exhaustive survey of investigations of ionic equilibrium of fluorescein and its derivatives is presented in [5] where the electronic absorption spectra of ionic and molecular species and constants of step dissociation of fluorone dyes in true and organized solutions are presented. From the entire series of fluorone dyes, unsubstituted fluorescein has been studied in most ample details [6–13]. The ionic equilibrium of halogenated derivatives of fluorescein (eosin Y and erythrosin B) has less been studied. A correct definition of the dissociation constants of eosin Y and erythrosine B is complicated by the spectral proximity of absorption line profiles of dianions and anions as well as by the limited solubility of their neutral species. The time-resolved and steady-state spectral-luminescent properties of ionic states have also been most thoroughly investigated for fluorescein having the quantum yield (0.92 [14]) and fluorescence lifetime (4.1 ns [6]) maximum for dianion fluorone dyes. It is well known that dianions of halogenated fluoresceins have much lower quantum yield and fluorescence lifetime: 0.2 and 1.43 ns for eosin Y and 0.02 and 0.12 ns for erythrosin B, respectively [14]. The spin-orbital interaction in dyes comprising heavy atoms (bromine or iodine) significantly reduces the probability of emission processes thereby complicating the time-resolved study of ionic species of eosin Y and erythrosin B. A comparative investigation of spectral properties of ionic states of the series of fluorone dyes is lacking in the literature. Combined application of absorption and fluorescence (including polarized and time-resolved) measurements allows qualitative information to be obtained on the ionic equilibriums of dyes without processing of large array of spectral data with minimum step in pH.
Siberian Federal University, Krasnoyarsk, Russia, e-mail:
[email protected];
[email protected]. Translated from Izvestiya Vysshikh Uchebnykh Zavedenii, Fizika, No. 12, pp. 48–54, December, 2013. Original article submitted August 12, 2013. 1370
1064-8887/14/5612-1370 2014 Springer Science+Business Media New York
Fig. 1. Ionic species of fluorone dyes: fluorescein (R = H), eosin Y (R = Br), and erythrosin B (R = I).
MATERIALS AND METHODS In this work, we used sodium salts of fluorescein (C20H10O5Na2), eosin Y (C20H6Br4O5Na2), and erythrosin B (C20H6I4O5Na2) produced by Fluka and Sigma Chemicals. To prepare aqueous solutions of dyes with pH changing in wide limits, distilled water, aqueous solutions of KOH alkali, and hydrochloric acid were used. The dye concentration in solutions was 8 µM. Measurements were performed 30–40 min after preparation of solutions. The pH value of solutions was measured with an HI 2210 pH-meter (Hanna Instruments, the USA). The absorption spectra were recorded with a Lambda 35 spectrophotometer (PerkinElmer, the USA). The fluorescence spectra and decay were measured on a Fluorolog 3-22 spectrofluorimeter (Horiba Jobin Yvon, the USA) with the possibility of lifetime measurements. The luminescence spectra of fluorescein were excited at a wavelength of 470 nm, of eosin Y at 470 nm, and of erythrosin B at 480 nm. The spectra obtained were corrected for the reabsorption and sensitivity of the recording system. The relative quantum fluorescence yield was calculated as a ratio of the area under the emission spectrum to the relative portion of light absorbed at the excitation wavelength. The fluorescence polarization was measured in the emission band maximum. A flash-lamp (150 W) with pulse duration of 3 µs was used to measure the delayed fluorescence spectra. The delay time was 50 µs, and the signal of 20 pulses was accumulated. The fluorescence decay was measured in the emission band maximum upon excitation by laser light-emitting diode NanoLED (λmax 453 nm and 50 nm) with pulse duration of about 1.3 ns. The deconvolution procedure and the analysis of the fluorescence decay were performed using the special DAS6 program. Standard quartz cells with cross sections of 10 10 mm were used to investigate solutions for L-geometry of excitation. Measurements were performed at room temperature. 1371
Fig. 2. Absorption cross sections of the fluorone dyes in aqueous solutions with the indicated pH values: fluorescein (a), eosin Y (b), and erythrosin B (c).
RESULTS AND DISCUSSION Absorption Spectra of Fluorone Dye in Aqueous Solution The character of changes of the fluorone dye absorption spectra accompanying variations of pH values (Fig. 2) is in good agreement with the data published in the literature. In basic solvent for fluorescein (Fig. 2a), the dianion species with maximum at a wavelength of 490 nm and bending of the curve near 475 nm are formed. The relative fraction of dianions decreases with pH reduction and simultaneous increase in the fraction of other protolythic species – anion (with two maxima near 474 and 453 nm), neutral species comprising three tautomers (zwitterion 22%, quinoid 11%, and lactone 67% [5]), and cation (with maximum at 437 nm). It is well known [6, 11] that fluorescein anion exists mainly as anion I (Fig. 1). The dianion species of eosin Y and erythrosin B also have intensive absorption bands with maxima at wavelengths of 517 nm for eosin Y and 526 nm for erythrosin B (Fig. 2b and c). The characters of change of the eosin Y and erythrosin B spectra with decreasing pH are in many respects similar: the amplitude of the spectrum decreases with decreasing pH, and the bathochromic shift is observed. This is due to an increase in the relative fraction of anions whose spectrum is red shifted and has smaller amplitude in comparison with the dianion species. It is well known that more than 80% of eosin anions exist as anion II species (Fig. 1) [5]. For low pH indices, eosin Y and erythrosin B are transformed into the neutral species with a wide and low-intensive absorption line profile. The neutral species are presented mainly as lactone (64%) and quinoid (31%) [5]. Thus, the character of change of the absorption spectra accompanying the pH variation of fluorescein and its halogenated derivatives differs qualitatively. The change of the amplitude of the absorption spectrum can serve as a quantitative characteristic of the decrease of the relative fraction of dianions with decreasing pH. From Fig. 3 it can be seen that for all examined dyes this dependence on pH is described by an S-shaped curve vanishing at low pH values. Fluorescence and Delayed Fluorescence of Fluorone Dye in Aqueous Solution Figure 4 shows the normalized fluorescence spectra of fluorone dyes for the indicated pH values of the solvent. The fluorescein line profile is broadened with decreasing pH value of the solvent, and in addition to the maximum, a bend arises at 545–555 nm. For eosin Y, the line profile remains similar with the maximum at 536 nm approximately up to pH = 5; then a shoulder arises at 585 nm and a bathochromic shift of the maximum up to 18 nm. For erythrosin B at pH > 5, the emission line profile with maximum at 547 nm remains unchanged, the additional shoulder also arises
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Fig. 3. Relative change of the optical density in the maximum of the fluorone dye absorption spectrum versus pH of the fluorescein (curve 1), eosin Y (curve 2), and erythrosin B (curve 3) solutions.
Fig. 4. Fluorescence spectra of the fluorone dyes in aqueous solutions with the indicated pH values: fluorescein (a), eosin Y (b), and erythrosin B (c).
near 600 nm, and the red shift of the maximum up to 32 nm is observed. The presence of the bend and shift of the maximum are due to the contribution of the anion or neutral species, whose quantum yield is smaller than that of dianion species, to the emission [11]. The fluorescence intensity decreases significantly with decreasing pH of the medium for all examined dyes. This is caused by the change of the portion of energy absorbed by different ionic states and their different quantum yields. The relative fluorescence quantum yield of dyes is shown in Fig. 5. It should be noted that the absolute value of the quantum yield in the series of dyes fluorescein – eosin Y – erythrosin B decreases significantly because of strengthening of the spin-orbital interaction caused by the presence of heavy atoms [14, 15]. According to [14], the range of variation of the absolute quantum fluorescein yield is 0.23–0.92 for a wide interval of pH indices. A nonzero minimum value is caused by the presence of fluorescent quinoid state [11]. For eosin Y this range is 0.004–0.2, and it is 0.0005–0.02 for erythrosin B. Values close to zero belong to non-fluorescent lactone or quinone neutral species. Investigation of the delayed fluorescence spectra upon pulsed excitation demonstrated that the character of change of the relative quantum yield versus pH is similar to the change of the quantum yield of the prompt fluorescence for eosin Y and erythrosin B. Since the observed delayed fluorescence belongs to the E-type, its intensity depends on the population density of the triplet dye levels and the probability of reverse intersystem crossing to the first singlet
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Fig. 5
Fig. 6
Fig. 5. Relative change of the fluorescence quantum yield of fluorone dyes vs. the pH value of the solution. Here curve 1 is for fluorescein, curve 2 is for eosin Y, and curve 3 is for erythrosin В. Fig. 6. Dependence of the fluorescence lifetime of fluorone dyes on the pH value. Here curve 1 is for fluorescein, curve 2 is for eosin Y, and curve 3 is for erythrosin B.
state. It seems most likely that these parameters are not too perturbed by the dye transition to the anion and neutral states. For fluorescein, we failed to register a significant signal of the delayed fluorescence because of the low yield to the triplet state [16].
Fluorescence Lifetime An analysis of the fluorescence decay of the fluorescein and eosin Y demonstrated that it can be described by a sum of two exponents with different lifetimes. Depending on the pH, the amplitudes of contributions of these components change. Below we used the lifetime obtained by averaging of two times considering their amplitude contributions. The average fluorescence lifetime of fluorone dyes depending on pH is shown in Fig. 6. The maximum fluorescein (4.1 ns) and eosin Y (1.28 ns) lifetimes belong to the dianion species. The average fluorescence lifetime decreases to 2.7 ns for fluorescein and 1.11 ns for eosin Y with decreasing pH. The measured lifetime of dianions was 0.14 ns for pH 11.73 for erythrosin B and 0.11 ns for pH 4.0, which is close to the limiting resolution of the experimental setup. In this case, the values obtained are in agreement with the data presented in [14].
Degree of Fluorescence Polarization The measured degrees of fluorescence polarization (P) are presented in Table 1. In low viscous solutions the P value provides information on the rotational relaxation of the direction of the dipole moment for the transition during the lifetime of the excited state and can be described by the Perrin formula [17]
P0 k T 1 3 P0 fl B , P 3V
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(1)
TABLE 1. Degree of Fluorescence Polarization and Effective Hydrodynamic Diameter of Fluorone Dyes for the Indicated pH Values of the Solvent Dye
pH = 2.3
pH = 5.1
pH = 11.7
P
D, nm
P
D, nm
P
D, nm
Fluorescein
0.016 0.001
0.74 0.03
0.009 0.001
0.73 0.04
0.007 0.001
0.86 0.04
Eosin Y
0.072 0.004
0.91 0.05
0.044 0.002
0.92 0.05
0.042 0.002
1.06 0.07
Erythrosin B
0.304 0.003
1.0 0.3
0.296 0.002
1.0 0.3
0.295 0.002
1.1 0.2
TABLE 2. Constants of Ionic Equilibrium of Fluorone Dyes Dye
From the absorption From the spectra quantum yield pKа pKа*
From the fluorescence lifetime pKа*
Fluorescein
6.18 0.05
6.06 0.06
5.54 0.12
Eosin Y Erythrosin B
3.10 0.05 3.74 0.06
2.97 0.04 3.54 0.05
3.32 0.15 –
Data presented in the literature pKa1 6.36–6.7 [6, 7, 12] 3.31–3.64 [5] 4.0–5.16 [5]
pKa2 4.31–4.4 [6, 7, 12] 2.48–2.77 [5] 3.79–3.92 [5]
where P0 is the limiting polarization degree (equal to 0.5 for liquid solutions), V is the volume of the molecule, T is temperature, is the dynamic viscosity of the medium, kB is the Boltzmann constant, and fl is the fluorescence lifetime. In calculation, values of the fluorescent lifetime measured in the present work were used. From the data presented in Table 1 it follows that the polarization degree increases in the series fluorescein – eosin Y– erythrosin B for all pH values and depends on the pH index for fluorescein and eosin Y. These changes can be caused by both different fluorescence lifetimes and different volumes of the molecules. The hydrodynamic diameter D calculated from formula (1) (in the approximation of a spherical molecule) has two tendencies: it increases from fluorescein to erythrosin B due to the increased bond lengths (С–H, C–Br, and C–I) in the process of halogen substitution [18] as well as increased size of the molecule in the alkaline medium. The last tendency can demonstrate that dianions preserve a larger solvate shell than other ionic states. For erythrosin B this tendency cannot be traced because of the large error caused by the low value of the fluorescence lifetime. Calculation of the Ionic Equilibrium Constants Rigorous calculation of the dissociation constants is connected with obtaining and processing of a large array of spectral data with minimum step in pH. Moreover, additional problem arises for eosin Y and erythrosin B connected with the proximity of the profiles and positions of dianion and anion lines, the latter of which cannot be experimentally measured in its pure form. In the present work, to calculate pKа of fluorone dyes, we used the approximate method based on the analysis of amplitude changes of the line profile for the dianion absorption maximum (see Fig. 3). The results obtained are presented in Table 2. The abscissa (pKа) of the bend point of this dependence on pH should be considered as the effective dissociation constant. More exact values of pKa2 can be found if we decompose the line profiles into the dianion and anion components and take into account the change of amplitudes of the dianion profile alone. In [13] we used this method based on the position of the pure ionic states presented in [8]. After the decomposition procedure, we took into account only the amplitude changes of the line profile for the dianion species. The pKа2 value thus obtained was 6.3. Due to the different quantum yields and lifetimes of ionic species, the value of the dissociation constant (pKа*) can be found from the abscissa of the bend point of the S-shaped quantum yield function and the fluorescence lifetime depending on pH (see Figs. 5 and 6). It should be noted that in the present work, the method of estimation of the dissociation constant from the measured fluorescence lifetime has been suggested for the first time. 1375
In [7, 11, 12, 19] it was indicated that the dissociation constants of the excited states differed from those of the ground states. Moreover, pKа* and pKа correspond to each other when the ionic equilibrium in the excited state is established much more slowly than the emission process. In all other cases, the pKа* value differs from the corresponding value for the ground state. According to our measurements, pKа derived from the absorption spectra and pKа* derived from the measured quantum yields were close in values. CONCLUSIONS The correct determination of the multistage dissociation constants for dyes is possible only with allowance for a number of factors: simultaneous changes of positions of the acid-base and tautomeric equilibriums, ionic force of the solvent, influence of the concentration on dimerization and solubility of some ionic species, and chemical purity of dyes and solvent. Therefore, even with large arrays of pH values obtained in a wide spectral range, a wide variability of the dissociation constants can be found in the literature. We have demonstrated that the character of changes of the absorption spectra accompanying the pH change differs significantly for fluorescein and its halogenated derivatives caused by different structures of their anion species known from the literature. Despite of this, the pKа values found from the change of the amplitude of the absorption spectrum for all three dyes lie in the range corresponding to dianion-anion and anion-neutral equilibrium constants and can be considered as the dianion-anion equilibrium constant estimated from below. The fluorescent properties of fluorescein and its halogenated derivatives differ as well. For low pH values, fluorescein has nonzero quantum yield caused by the presence of fluorescent quinoid neutral species. Since the change of the quantum yield is caused by redistribution of the relative fraction of fluorescent dianion, anion, and neutral species, the values obtained have the meaning of the effective equilibrium constant (the dianion-anion equilibrium constant for eosin Y and erythrosin B estimated from below). In our measurements, pKа derived from the absorption spectra and pKа* derived from measurement of the quantum yields were close in values, that demonstrated a high emission rate in comparison with the rate of establishing the ionic equilibrium. The polarization fluorescence measurements used to estimate the size of molecules demonstrated the increase of the hydrodynamic diameter in the series fluorescein – eosin Y– erythrosin B caused by the increase of the С–H, C– Br, and C–I bond lengths as well as by the maximal solvate shell of dianions of the dyes.
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