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Complex fluorescence decay of quinine bisulphate in aqueous sulphuric acid solution
CHEMICALPHYSICSLElTERS
Volume 88, number 1
COMPLEX FLUORESCENCE DECAY OF QUITE IN AQUEDUS SULP~~IC
23 April 1982
R~S~P~ATE
ACID SOL~IDN
Rccc~ved I3 October 1981;in finalform I9 February1982 l%~orcsccnce of quinme txsulphate ISshown to be two-component. In 1.0 N H2S04 solutron the major component has a dccq time =20 IX, but there USAminor component with decay tune -2 ns with a dtffcrcnt tluorcsccna: spectrum_ it is rc. commended tbst the compound NOI be used as a standard for ~euy-t~mc m~surements
been used to measure the ~~orescence yield of many flwrophores [?-I. Consequently the photophysical
1. introduction
Quinine blsuiphate (QBS) (I) dissokd m I .O N H,SO4 ISa commonly used standard for fluorescence measurements. The absolute fluorescencespectrum of this system IS frequently used to correct for the wnvelength dependence of lurn~~e~~nce detectIon systems I
[I] and the absolute fluorescence quantum yield has
properties of QBS in I .O N H$Od have been the sub” Ject of extensive mvestlgations.As a result of these a number of unusual effects have been discovered, which m&de: IIsubstantial disagreementbetween the radiative ljfetune measured from quantum yield and decay time data and that calculated from the abso~tion spectrum [3] ; a dependence af the putrescence quantum yield on the normalrty of the acid used as solvent [4] and fiially a red-shift in the emissionas the excitation wavelengthis changed between 340 and 420 nm [5]. These edge excitation effects are quite different from those observed for other fluorophores [6] since they occur m llwd media.
T&&z1 Fluorcsccncc decay t;mcs of qumine bIsuiphutc satut~on
Con~ntra~on
emission (nm)
7 (IIs)
A~a~ytiu~ method
0.1 N H2S04
to-’
IO* -
total total total total
19.4 a) 20bl 19.4 c) 188d)
method af momeats metbod of moments log plot
l0-2
470 k&I
193e) 0
Fourier least-squares
0-t N &SO4 0 I N H2S0.1
0.1 N HzS04 t NHzS04 1 N H2SOj
‘I Ref. {18]. ‘) Ref. 120). @Ref. 1211. c) Ref. [22]. fl Rcf 123] , these authors were unable to fit the decay wetf to a single component.
a)Ref. [19).
22
0 009.26141’82fOOOO-0000~$02.75 0 1982 Nosh-Homed
Volume
88, number 1
Cl~E~lrCALPHYSICSLClTERS
Despite these complexities, the compound has been recommended for use as a standard for the measurement of fluorescence decay times, and fable 1 shows the values of the single-component decay measured with the techmque used for analysis. In all cases, total fluorescence was monitored. In view of these complexitIes we have measured the fluorescence decay of QBS in 1.O N H2SO4 as a function of the emission wavelength monitored.
23 Aprd 1982
three times from water and stored rn a dessrcator. The corrected emission spectrum (not shown here) was m excellent agreement with that gtven rn the hteraturc 111. Water was doubly or triply distr!Ied and sulphurlc acid was BDH “Aristar” grade. All solvents were essentially non-fiuorescent on the maxfmum sensttrvity scale of an hfPF-4 fluorrmeter. Concentrations of QBS were in the region of 5 X 10q6 M.
3 Results and discussions 2. Exper~entai Fluorescence decay measurements were made usmg a synchronously pumped-mode-locked-caanty-dumped dye laser as the excitation source. The pulse repetttlon rate was 4 MHz in all experiments. Full details of this system wtll be given elsewhere 17). The output of the rhod~ine 6G dye laser was frequency doubled m an ADP crystal and the resulting W radiation directed mto the sample cell. The fluorescence was spectrally dispersed by a Rank-Precision D330 monochromator (with a resolution of=4 MI) and the fluorescence decay collected usmg conventional stngle photon countmg techniques [S] _All ~uore~ence decays had 30000 counts in the channel of m~um counts. These decays were analysed by the method of non&near ieastsquares iterative reconvolution [9] and the “‘goodness of fit” judged by the distribution of the residual and autocorrelation of residual plots [IO] and the value of the reduced chi-square (xt) [II]. The instrument response function (full width at half ma~um height ~600 ps) was measured at the excitation wavelength (=3 IO run). No correction was made for the wavelengthdependent time response of the photomultiplier tube [12,13] ; random residual plots suggest that a correction for this effect was unnecessary for these data. Time-resolved emission spectra were obtatned by setting time gates in the multichannel analyser, operated in the multichanne1 scaling mode, and scanning the emission monochromator [14]. The time-resolved spectra presented are the sum of several runs, which corrected for any small dnfts in the intensity of the laser system. These were collected to =15000 counts m the maximum. The spectra presented are not corrected for the wavelength response of the detector systern. QW.ne bisulphate (I’& B) was recrystalbsed two or
The wavelength dependence of the fluorescence decay of QBS m I 0 N H2S04 is &own m tabh I and fig. I _The fluorescence decay parameters arc obtamcd in the least-squares method used here from the equatron I
I(I)
=/P(f)
G(r
-
I’) dr’,
0
where I(r) is the experuncntally measured decay function, P(f) IS the instrument response function (measured at the excitation wavelength) and G(r) is the function which represents the response of the system to a pulse excttation. Two functions of G(t) have been used here. A smgle exponentlai function clearly fails to descrtbe the observed decay, non-random rcsrdual and autocorrelatlon plots (fig. 2;3) and xz values
Fg. 1. Wnvclcngth-dcpcndcnrfluoresccnccdcwys of QBSIII 10 N H_LSOJ(3) instrument responsefunction (b) l3uorescence decay at 425 nm. Sohd lute is the recon~olweddecay (dual eupcncntial fit) and dots rcprcsentoriginsf decay data k) Fluorescencedecay at 52.5 nm, presented 3s above Channel width = 0.164 ns/chrnncl
23
Volume 88, number 1
-102 ’
120
23 Apd 1982
CHEWICALPHYSICSLETTERS
> 1.8 (table 7). at all wavelengths.However the data are well fit by a sum of two exponentials (xi values LO-I.3 and random residual and autocorrelation of residual plots - table 2 and fig. 2b). The bfetune data clearly show a two-component fluorescence decay on the bhre edge of the emission with a short (=2 ns) decay and a longer (~20 ns) decay, which is dominant, the short decay contributmg ~2% of the total fluorescence at 450 nm (table 1). On the red edge of the emission a fluorescence risetlme
240 Pvm.9.u*1)ccq
(~2 ns) is observed which may also be seen in the log plot of the 550 run decay (fig. 1). Againthe long-~ved
(31
(==20.7ns) decay is dominant (*98(r)). Previousdecay measurements 13,151have reported only a single lifetime for quinine in sulphurrcacid solution, with a lifetime around 20 ns. We can only resolve the small sec-
,;_-___:
ond component because of the greater intensity and improved tune resolution available with laser excltation sources. These allow waveIength re~~ution of the fluorescence decay and rmproved signal-to-noise ratios, with a consequent increase in the confidence one may
-331
120
2LO cHul”CL hwem
360
have in deconvolution procedures, which are important in the study of short lifetimes. Usingflashlampexcitation and observing the whole
L&J
Ib)
Fig. 2. (a) Residul plots from ~ngle~omponent lits. (I) 425 nm, (II) 525 nm. @) Residual plots from two~omponent fits (I) 425 nm (it) 525 nm
of the emission band we are unable to resolve a short component, although the fit to a singleexponential function is not good (x: > 2). Interestmgly this fit is improved when the excitation from the flashlamp ts
Table 2 Fluoresccncc decay of QBS in 1 N HaSO fitted to smgle and double exponential limcttons Frgures in parentheses represent 3 times standard devlatlon G(i) a) &T
A, cmfh f At e+Z
a) See teut.
24
h (nm)
Al
rt (ns)
A2 Z*)
400 450 500
17.84(0.2) 19.73(0.01) 20.63(0 05)
-
550
20~94(0.005)
-
400 425 450 500 525
0.54 0 30 0.13 -0.12 -0.19
2 6rXO.12) 258(0.21) 3.97(0.75) 1.61(0 50) 1.74(0.30)
550 575
-0.21 -0.25
2.16(0.24) 1.92(0.36)
-
1 %
At fl At ‘t +A2 72
32.0 3.5
-
21.8
-
3.2
-
1.03 0.99 1.36 1.52
l9.i4(0_03~ 19 09(0 09) 20.17(0.09) 205 l(O.06)
1.1 1.1 1.3 I .2
0.01 0.04 002 001
157 1.60 1.60
2052(0 06) 20.68(0 06) 20 66(0.~)
1.O 1.3 1.2
0.01 0.01 0.02
Volume88, number 1
CHEMICAL PHYSICS LETTERS
23 April 1982
*365 MI, which givesa hfetrme of 20.3 ns, in quite good agreement with ref. 131,with a x: value of 1.3. We do not believe that the duat component, wave-
shift ISobserved when the time gate is moved from 0 to 46.0 ns after the initial excitation. This shift ISnot
iength~ependent, fluorescence obtained can be due to any artefact since a good s&-&e exponential decay
shape. ale-resoIved
was obtained for 2 ~inopyridine dissolvedm 1.O N H-$50, [16) under identical condittons to those used for the QBSmeasurements. We have also obtained the time-resolved emission spectra of QBS in H2S04 shown in fig. 3. A small red-
torttons due to the convolution of the fluorescence decay with the excitation pulse [14,16] but the shift observed here is larger than that observed due to this artefact [ 14,171. There have been numerous reports [4,18] of the decrease in quantum yteld of QBS as the normality of the acid ISchanged and we are able to repeat these observations. We have also measured the wavelength dependence of the fluorescence decay of QBS m 0.1 N H2SO4 under the same conditions that were used for the 1.ON system. The data are presented in table 3. It ISclear that a double exponential function is also required to descrrbe these data, but in this case we are unable to resolve a risetbne on the red edge of the emission. Also the errors in rI become rather large at the longer wavelengths. It ISnoticable that the lifetime of the longer-lived species is sigmftcantly shorter than that obtamed from the 1.O N samples and approximately reflects the decrease m quantum yield observed when the acid concentration is changed. It seems however that the decay routes of QBS in 1 N and 0.1 N H2S04 solution are also different. There are several possibilities which could account for the complex decay characteristics observed, mclud-
IO
a20
37s
510
465
555
1nrnl WAVELENGTH Frg. 3. Uncorrected ume resolved fluorescence spectra of QBS in 1.0 N &SO4 (a) Early gatcd spectrum, AI = 0 ns, ar = 1 8 ns; 0s) late grtcd spectrum. Al = 46 ns, ar = 2.9 ns - - Total fluorescence spectrum [recorded on the amc syslcm as
(a) and @Il.
accompanied by any signi~~nt
change in spectral spectra obtained by the direct method employed here are known to be subject to dis-
Table 3 Fluorescence deczy of QBS in 0.1 N Hz804 iitted to smgle and doubte e~ponent~l functions. rigures in parentheses represent 3 times standard deviation
e+lri
A,
a)Seetext.
&l
400 450 500 550 + A2
earls2
400 450 500 550b)
0.52 0.15 0.11 -
16.97(0.40) lB.74(0 40) 19.23(0 40) 19.99(0.40)
-
2 62(0.12) 3.63(0.70) 10.30(6.2)
1.02 1.35 1.34
31.6 3.3 16 3.5 18.17(0.08) 19.12(0.08) 19.74(0.54) -
1.3 I .2 1.3
bt Could not be fitted to a double exponential fun&on. 25
Volume
88, number 1
CHEMICAL
PHYSICS
ing the involvement of different conformers. These are currently under mvestigatlon, and will be reported elsewhere. We wish at this point merely to state that because the decays are complex, the use of qumine bisulphate as a fluorescence standard for decay-time measurements should be discouraged.
Acknowledgement We are
grateful to the Science and Engineering ReSociety for fiianclal sup-
23 Aprd
LETTERS
1982
[8] W.R Ware, m: Creation and dctectron of the ekcltcd slate. Vol. 1. cd. A.A. Lamola (Dekker, New York, 1971). [9] D.V. O’Connor, W R. Ware and 1 C. Andre. J. Phys Chem 83 (1979) 1333. (LO] A. Grinwld and LZ. Steinberg, Anal. Biochcm. 59 (1974) 583. P.R. Bevingtan. Data reduction and errar analysts for the physIca sciences (McGraw-Hdl. New York, 1969) [ 121 D hl Rayner. A.E. McKinnon and A G. Swbo, Rev. Scl Instr. 48 (1977) 1050. [ 131 Ph. Wahl. J.C. Auchet and B. Donzel. Rev. Sci Instr. 45 (1974) 28.
[ 111
[ 141
port.
S R. Mecch, D.V. O’Connor. A J. Roberts and D. Philips. Photochem. Photobrol. 33 (1981) 159. [ 151 I B. Bertman, Handbook of fkrorescencc spectra of aromaLlc compounds (Andema Press. New York, 1965) [ 16j J H. Easter, R.P. De Toma and L. Brand. Biophys. J. 16 (1976)571.
References
[ 171
search Council and the Royal
[ 11 W.H. Mclhulsh, Appl Opt. 14 (1975) 26 121 J.N. Demasand G.A. Crosby. J. Phys. Chem. 75 (1971) 991. [3] J.B. Buks, Photophysin of aromatic mohxules (WdeyInterscience. New York, 1970) 14) W.R Dawson and M.\V. Wmdsor, J. Phys Chem (1968) 3251.
72
151 A N. Fletcher, J. Phys. Chem. 72 (1968) 2742 161 K I. llah and T. Azumi, J. Chem. Phys. 62 (1975) 3431. 171 D.V. O’Connor, AJ. Roberts, R A. Lmpert, S R. hfcech, L Chewter and D. Phdlips, Standards for Nanosecond Lrfetime hlersurcments. Anal. Chem , submttfed far publication.
26
K.P. Ghiggmo, A.G. Lee, S.R. hleech. D.V. O’Connor and D Phillips. Blochermstry. to be pubbshed. [lS] R F.Chen, J. Res. Natl Bur. Std. US 76A (1972) 593. [ 191I. Yguenblde. tn. Rfethods m enzymology, Vol. 26, cds C H-W. Hirs and S.N. Ttmasheff York, 1972). 1201 R. Schuyler 813.
(Aadcmic
and 1. Iscnberg, Rev Scl lnstr
Press. New 42 (1971)
1211 R Lopez-Delgado. A. Tnmer and I.H. hlunro, Chcm. Phy! 5 (1974) 72. [22j U P. Wdd. A.R. Holzwarth and H P. Good, Rev SCI Instr.48 (1977) 1621. 1231 C-H. Harris and B I;. Selinger, Australwn J. Chem 32 (1979) 2111.
Volume 88, number 1
COMPLEX FLUORESCENCE DECAY OF QUITE IN AQUEDUS SULP~~IC
23 April 1982
R~S~P~ATE
ACID SOL~IDN
Rccc~ved I3 October 1981;in finalform I9 February1982 l%~orcsccnce of quinme txsulphate ISshown to be two-component. In 1.0 N H2S04 solutron the major component has a dccq time =20 IX, but there USAminor component with decay tune -2 ns with a dtffcrcnt tluorcsccna: spectrum_ it is rc. commended tbst the compound NOI be used as a standard for ~euy-t~mc m~surements
been used to measure the ~~orescence yield of many flwrophores [?-I. Consequently the photophysical
1. introduction
Quinine blsuiphate (QBS) (I) dissokd m I .O N H,SO4 ISa commonly used standard for fluorescence measurements. The absolute fluorescencespectrum of this system IS frequently used to correct for the wnvelength dependence of lurn~~e~~nce detectIon systems I
[I] and the absolute fluorescence quantum yield has
properties of QBS in I .O N H$Od have been the sub” Ject of extensive mvestlgations.As a result of these a number of unusual effects have been discovered, which m&de: IIsubstantial disagreementbetween the radiative ljfetune measured from quantum yield and decay time data and that calculated from the abso~tion spectrum [3] ; a dependence af the putrescence quantum yield on the normalrty of the acid used as solvent [4] and fiially a red-shift in the emissionas the excitation wavelengthis changed between 340 and 420 nm [5]. These edge excitation effects are quite different from those observed for other fluorophores [6] since they occur m llwd media.
T&&z1 Fluorcsccncc decay t;mcs of qumine bIsuiphutc satut~on
Con~ntra~on
emission (nm)
7 (IIs)
A~a~ytiu~ method
0.1 N H2S04
to-’
IO* -
total total total total
19.4 a) 20bl 19.4 c) 188d)
method af momeats metbod of moments log plot
l0-2
470 k&I
193e) 0
Fourier least-squares
0-t N &SO4 0 I N H2S0.1
0.1 N HzS04 t NHzS04 1 N H2SOj
‘I Ref. {18]. ‘) Ref. 120). @Ref. 1211. c) Ref. [22]. fl Rcf 123] , these authors were unable to fit the decay wetf to a single component.
a)Ref. [19).
22
0 009.26141’82fOOOO-0000~$02.75 0 1982 Nosh-Homed
Volume
88, number 1
Cl~E~lrCALPHYSICSLClTERS
Despite these complexities, the compound has been recommended for use as a standard for the measurement of fluorescence decay times, and fable 1 shows the values of the single-component decay measured with the techmque used for analysis. In all cases, total fluorescence was monitored. In view of these complexitIes we have measured the fluorescence decay of QBS in 1.O N H2SO4 as a function of the emission wavelength monitored.
23 Aprd 1982
three times from water and stored rn a dessrcator. The corrected emission spectrum (not shown here) was m excellent agreement with that gtven rn the hteraturc 111. Water was doubly or triply distr!Ied and sulphurlc acid was BDH “Aristar” grade. All solvents were essentially non-fiuorescent on the maxfmum sensttrvity scale of an hfPF-4 fluorrmeter. Concentrations of QBS were in the region of 5 X 10q6 M.
3 Results and discussions 2. Exper~entai Fluorescence decay measurements were made usmg a synchronously pumped-mode-locked-caanty-dumped dye laser as the excitation source. The pulse repetttlon rate was 4 MHz in all experiments. Full details of this system wtll be given elsewhere 17). The output of the rhod~ine 6G dye laser was frequency doubled m an ADP crystal and the resulting W radiation directed mto the sample cell. The fluorescence was spectrally dispersed by a Rank-Precision D330 monochromator (with a resolution of=4 MI) and the fluorescence decay collected usmg conventional stngle photon countmg techniques [S] _All ~uore~ence decays had 30000 counts in the channel of m~um counts. These decays were analysed by the method of non&near ieastsquares iterative reconvolution [9] and the “‘goodness of fit” judged by the distribution of the residual and autocorrelation of residual plots [IO] and the value of the reduced chi-square (xt) [II]. The instrument response function (full width at half ma~um height ~600 ps) was measured at the excitation wavelength (=3 IO run). No correction was made for the wavelengthdependent time response of the photomultiplier tube [12,13] ; random residual plots suggest that a correction for this effect was unnecessary for these data. Time-resolved emission spectra were obtatned by setting time gates in the multichannel analyser, operated in the multichanne1 scaling mode, and scanning the emission monochromator [14]. The time-resolved spectra presented are the sum of several runs, which corrected for any small dnfts in the intensity of the laser system. These were collected to =15000 counts m the maximum. The spectra presented are not corrected for the wavelength response of the detector systern. QW.ne bisulphate (I’& B) was recrystalbsed two or
The wavelength dependence of the fluorescence decay of QBS m I 0 N H2S04 is &own m tabh I and fig. I _The fluorescence decay parameters arc obtamcd in the least-squares method used here from the equatron I
I(I)
=/P(f)
G(r
-
I’) dr’,
0
where I(r) is the experuncntally measured decay function, P(f) IS the instrument response function (measured at the excitation wavelength) and G(r) is the function which represents the response of the system to a pulse excttation. Two functions of G(t) have been used here. A smgle exponentlai function clearly fails to descrtbe the observed decay, non-random rcsrdual and autocorrelatlon plots (fig. 2;3) and xz values
Fg. 1. Wnvclcngth-dcpcndcnrfluoresccnccdcwys of QBSIII 10 N H_LSOJ(3) instrument responsefunction (b) l3uorescence decay at 425 nm. Sohd lute is the recon~olweddecay (dual eupcncntial fit) and dots rcprcsentoriginsf decay data k) Fluorescencedecay at 52.5 nm, presented 3s above Channel width = 0.164 ns/chrnncl
23
Volume 88, number 1
-102 ’
120
23 Apd 1982
CHEWICALPHYSICSLETTERS
> 1.8 (table 7). at all wavelengths.However the data are well fit by a sum of two exponentials (xi values LO-I.3 and random residual and autocorrelation of residual plots - table 2 and fig. 2b). The bfetune data clearly show a two-component fluorescence decay on the bhre edge of the emission with a short (=2 ns) decay and a longer (~20 ns) decay, which is dominant, the short decay contributmg ~2% of the total fluorescence at 450 nm (table 1). On the red edge of the emission a fluorescence risetlme
240 Pvm.9.u*1)ccq
(~2 ns) is observed which may also be seen in the log plot of the 550 run decay (fig. 1). Againthe long-~ved
(31
(==20.7ns) decay is dominant (*98(r)). Previousdecay measurements 13,151have reported only a single lifetime for quinine in sulphurrcacid solution, with a lifetime around 20 ns. We can only resolve the small sec-
,;_-___:
ond component because of the greater intensity and improved tune resolution available with laser excltation sources. These allow waveIength re~~ution of the fluorescence decay and rmproved signal-to-noise ratios, with a consequent increase in the confidence one may
-331
120
2LO cHul”CL hwem
360
have in deconvolution procedures, which are important in the study of short lifetimes. Usingflashlampexcitation and observing the whole
L&J
Ib)
Fig. 2. (a) Residul plots from ~ngle~omponent lits. (I) 425 nm, (II) 525 nm. @) Residual plots from two~omponent fits (I) 425 nm (it) 525 nm
of the emission band we are unable to resolve a short component, although the fit to a singleexponential function is not good (x: > 2). Interestmgly this fit is improved when the excitation from the flashlamp ts
Table 2 Fluoresccncc decay of QBS in 1 N HaSO fitted to smgle and double exponential limcttons Frgures in parentheses represent 3 times standard devlatlon G(i) a) &T
A, cmfh f At e+Z
a) See teut.
24
h (nm)
Al
rt (ns)
A2 Z*)
400 450 500
17.84(0.2) 19.73(0.01) 20.63(0 05)
-
550
20~94(0.005)
-
400 425 450 500 525
0.54 0 30 0.13 -0.12 -0.19
2 6rXO.12) 258(0.21) 3.97(0.75) 1.61(0 50) 1.74(0.30)
550 575
-0.21 -0.25
2.16(0.24) 1.92(0.36)
-
1 %
At fl At ‘t +A2 72
32.0 3.5
-
21.8
-
3.2
-
1.03 0.99 1.36 1.52
l9.i4(0_03~ 19 09(0 09) 20.17(0.09) 205 l(O.06)
1.1 1.1 1.3 I .2
0.01 0.04 002 001
157 1.60 1.60
2052(0 06) 20.68(0 06) 20 66(0.~)
1.O 1.3 1.2
0.01 0.01 0.02
Volume88, number 1
CHEMICAL PHYSICS LETTERS
23 April 1982
*365 MI, which givesa hfetrme of 20.3 ns, in quite good agreement with ref. 131,with a x: value of 1.3. We do not believe that the duat component, wave-
shift ISobserved when the time gate is moved from 0 to 46.0 ns after the initial excitation. This shift ISnot
iength~ependent, fluorescence obtained can be due to any artefact since a good s&-&e exponential decay
shape. ale-resoIved
was obtained for 2 ~inopyridine dissolvedm 1.O N H-$50, [16) under identical condittons to those used for the QBSmeasurements. We have also obtained the time-resolved emission spectra of QBS in H2S04 shown in fig. 3. A small red-
torttons due to the convolution of the fluorescence decay with the excitation pulse [14,16] but the shift observed here is larger than that observed due to this artefact [ 14,171. There have been numerous reports [4,18] of the decrease in quantum yteld of QBS as the normality of the acid ISchanged and we are able to repeat these observations. We have also measured the wavelength dependence of the fluorescence decay of QBS m 0.1 N H2SO4 under the same conditions that were used for the 1.ON system. The data are presented in table 3. It ISclear that a double exponential function is also required to descrrbe these data, but in this case we are unable to resolve a risetbne on the red edge of the emission. Also the errors in rI become rather large at the longer wavelengths. It ISnoticable that the lifetime of the longer-lived species is sigmftcantly shorter than that obtamed from the 1.O N samples and approximately reflects the decrease m quantum yield observed when the acid concentration is changed. It seems however that the decay routes of QBS in 1 N and 0.1 N H2S04 solution are also different. There are several possibilities which could account for the complex decay characteristics observed, mclud-
IO
a20
37s
510
465
555
1nrnl WAVELENGTH Frg. 3. Uncorrected ume resolved fluorescence spectra of QBS in 1.0 N &SO4 (a) Early gatcd spectrum, AI = 0 ns, ar = 1 8 ns; 0s) late grtcd spectrum. Al = 46 ns, ar = 2.9 ns - - Total fluorescence spectrum [recorded on the amc syslcm as
(a) and @Il.
accompanied by any signi~~nt
change in spectral spectra obtained by the direct method employed here are known to be subject to dis-
Table 3 Fluorescence deczy of QBS in 0.1 N Hz804 iitted to smgle and doubte e~ponent~l functions. rigures in parentheses represent 3 times standard deviation
e+lri
A,
a)Seetext.
&l
400 450 500 550 + A2
earls2
400 450 500 550b)
0.52 0.15 0.11 -
16.97(0.40) lB.74(0 40) 19.23(0 40) 19.99(0.40)
-
2 62(0.12) 3.63(0.70) 10.30(6.2)
1.02 1.35 1.34
31.6 3.3 16 3.5 18.17(0.08) 19.12(0.08) 19.74(0.54) -
1.3 I .2 1.3
bt Could not be fitted to a double exponential fun&on. 25
Volume
88, number 1
CHEMICAL
PHYSICS
ing the involvement of different conformers. These are currently under mvestigatlon, and will be reported elsewhere. We wish at this point merely to state that because the decays are complex, the use of qumine bisulphate as a fluorescence standard for decay-time measurements should be discouraged.
Acknowledgement We are
grateful to the Science and Engineering ReSociety for fiianclal sup-
23 Aprd
LETTERS
1982
[8] W.R Ware, m: Creation and dctectron of the ekcltcd slate. Vol. 1. cd. A.A. Lamola (Dekker, New York, 1971). [9] D.V. O’Connor, W R. Ware and 1 C. Andre. J. Phys Chem 83 (1979) 1333. (LO] A. Grinwld and LZ. Steinberg, Anal. Biochcm. 59 (1974) 583. P.R. Bevingtan. Data reduction and errar analysts for the physIca sciences (McGraw-Hdl. New York, 1969) [ 121 D hl Rayner. A.E. McKinnon and A G. Swbo, Rev. Scl Instr. 48 (1977) 1050. [ 131 Ph. Wahl. J.C. Auchet and B. Donzel. Rev. Sci Instr. 45 (1974) 28.
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