R
− in alkaline solution has a relatively intense fluorescence emission spectrum with a maximum at 604 nm, while the RH
fluorescence is much broader and weaker with a maximum at 612 nm and a second, shorter wavelength peak around 565 nm
(Fig. 2). RH in S2 and S4 (not shown) fluoresces over a similar range to RH in solution (Fig. 2A) and exhibits a single
well-resolved maximum at ~573 nm. This is blue shifted from that of the dye in solution, although the latter is very noisy
due to extremely low emission intensity. The emission from RH in S2 is an order of magnitude more intense than that in
solution. Addition of TX-100 to the sol-gel matrix (S3) has little effect on the steady state emission of RH.
As pH increases the emission from sol-gel immobilised resorufin becomes more intense. Immobilisation of the dye in pure
sol-gel (S2) causes a hypsochromic shift of 15 nm in the emission spectrum of R
− (Fig. 2B). In the S3 sol-gel, the addition
of the TX-100 causes a bathochromic shift (relative to S2) and R
- emission in S3 is practically identical to the solution case.
3.2
Fluorescence lifetimes:
The fluorescence lifetime of R
−
, in 0.1 M phosphate buffer at pH 12.2, is a single exponential with a lifetime of 3.3 (550
nm) or 3.4 (600 & 650 nm) ns depending on the emission wavelength. At low pH
¥ (~2.6) the situation is more complex,
with there being a marked dependence on the emission wavelength at which the lifetime is measured. At an emission
wavelength of 550 nm the decay is a single exponential with a lifetime of 0.3 ns, while at longer wavelengths the decay
becomes bi-exponential with an average lifetime of 0.67 ns at 600 nm and 0.55 ns at 650 nm, indicating the presence of
some unprotonated anion. Plotting this lifetime change over the pH range 2.6 to 12.2 (Fig. 3) shows that the decay changes
from a bi-exponential to a single exponential above pH 8, as the contribution of the protonated species diminishes.
In fitting this experimental data, the short lifetime (τ 1 due to RH) was kept constant at 0.3 ns while the remaining parameters
(relative amplitudes and τ 2 ) were allowed to vary. At the low pH between 2.6 and 4.0, the lifetime (τ 2 ) of the longer-lived
component undergoes an initial sharp increase, before remaining relatively constant up to pH 8. Above pH 8, the decay fits
to a single exponential, indicating that R
− is the sole emitting species, and fluorescence from RH is no longer a contributing
factor. Resorufin exhibits similar decay characteristics at 632 and 650 nm to those observed at 600 nm with the decays
being bi-exponential below pH 8, and mono-exponential above pH 8. The relative amplitudes of each decay component
also vary in a similar manner to those recorded at 600 nm. In each case, the change in average lifetime (
τ
) with pH is
similar, with an increase of ~3 ns over the pH range 2.6 – 9.
15
The decay characteristics of resorufin in 0.1M phosphate buffer are somewhat different at 550 nm to the other wavelengths
investigated. There is a greater contribution from RH at higher pH values. For example, at pH 8.1, at emission wavelength
550 nm, the contribution from RH, accounts for 30% of the overall decay, while at the other wavelengths the contribution is
negligible. This is to be expected, as examination of the steady state emission spectrum (Fig. 2) shows the fluorescence
intensity of R
− is extremely weak at 550 nm in comparison to that at 600, 632, and 650 nm. As a result, at 550 nm
τ
shows
greater sensitivity at higher pH values (Fig. 3). Again, there is an increase of ~ 3 ns in
τ
between pH 2.6 and 10, but over
80% of this change (2.6 ns) occurs over the 5 – 8 pH range. Unfortunately, the pH response is best over the 5 – 6 range,
with only 1.3 ns change in
τ
between pH 6 and 8. The fluorescence lifetime of phosphate buffered solutions of resorufin
were also found to be unaffected by the presence of dissolved oxygen, chloride, or changes in buffer molarity (0.05 to 0.15
M) over the pH range 5.5 to 8.5.
¥ The low fluorescence intensity of RH at pH < 2.6 prevented the measurement of accurate lifetimes.
− in alkaline solution has a relatively intense fluorescence emission spectrum with a maximum at 604 nm, while the RH
fluorescence is much broader and weaker with a maximum at 612 nm and a second, shorter wavelength peak around 565 nm
(Fig. 2). RH in S2 and S4 (not shown) fluoresces over a similar range to RH in solution (Fig. 2A) and exhibits a single
well-resolved maximum at ~573 nm. This is blue shifted from that of the dye in solution, although the latter is very noisy
due to extremely low emission intensity. The emission from RH in S2 is an order of magnitude more intense than that in
solution. Addition of TX-100 to the sol-gel matrix (S3) has little effect on the steady state emission of RH.
As pH increases the emission from sol-gel immobilised resorufin becomes more intense. Immobilisation of the dye in pure
sol-gel (S2) causes a hypsochromic shift of 15 nm in the emission spectrum of R
− (Fig. 2B). In the S3 sol-gel, the addition
of the TX-100 causes a bathochromic shift (relative to S2) and R
- emission in S3 is practically identical to the solution case.
3.2
Fluorescence lifetimes:
The fluorescence lifetime of R
−
, in 0.1 M phosphate buffer at pH 12.2, is a single exponential with a lifetime of 3.3 (550
nm) or 3.4 (600 & 650 nm) ns depending on the emission wavelength. At low pH
¥ (~2.6) the situation is more complex,
with there being a marked dependence on the emission wavelength at which the lifetime is measured. At an emission
wavelength of 550 nm the decay is a single exponential with a lifetime of 0.3 ns, while at longer wavelengths the decay
becomes bi-exponential with an average lifetime of 0.67 ns at 600 nm and 0.55 ns at 650 nm, indicating the presence of
some unprotonated anion. Plotting this lifetime change over the pH range 2.6 to 12.2 (Fig. 3) shows that the decay changes
from a bi-exponential to a single exponential above pH 8, as the contribution of the protonated species diminishes.
In fitting this experimental data, the short lifetime (τ 1 due to RH) was kept constant at 0.3 ns while the remaining parameters
(relative amplitudes and τ 2 ) were allowed to vary. At the low pH between 2.6 and 4.0, the lifetime (τ 2 ) of the longer-lived
component undergoes an initial sharp increase, before remaining relatively constant up to pH 8. Above pH 8, the decay fits
to a single exponential, indicating that R
− is the sole emitting species, and fluorescence from RH is no longer a contributing
factor. Resorufin exhibits similar decay characteristics at 632 and 650 nm to those observed at 600 nm with the decays
being bi-exponential below pH 8, and mono-exponential above pH 8. The relative amplitudes of each decay component
also vary in a similar manner to those recorded at 600 nm. In each case, the change in average lifetime (
τ
) with pH is
similar, with an increase of ~3 ns over the pH range 2.6 – 9.
15
The decay characteristics of resorufin in 0.1M phosphate buffer are somewhat different at 550 nm to the other wavelengths
investigated. There is a greater contribution from RH at higher pH values. For example, at pH 8.1, at emission wavelength
550 nm, the contribution from RH, accounts for 30% of the overall decay, while at the other wavelengths the contribution is
negligible. This is to be expected, as examination of the steady state emission spectrum (Fig. 2) shows the fluorescence
intensity of R
− is extremely weak at 550 nm in comparison to that at 600, 632, and 650 nm. As a result, at 550 nm
τ
shows
greater sensitivity at higher pH values (Fig. 3). Again, there is an increase of ~ 3 ns in
τ
between pH 2.6 and 10, but over
80% of this change (2.6 ns) occurs over the 5 – 8 pH range. Unfortunately, the pH response is best over the 5 – 6 range,
with only 1.3 ns change in
τ
between pH 6 and 8. The fluorescence lifetime of phosphate buffered solutions of resorufin
were also found to be unaffected by the presence of dissolved oxygen, chloride, or changes in buffer molarity (0.05 to 0.15
M) over the pH range 5.5 to 8.5.
¥ The low fluorescence intensity of RH at pH < 2.6 prevented the measurement of accurate lifetimes.