The Rate Constant for Fluorescence Quenching
Michael W. Legenza and Charles J. Marzzacco Department of Physical Science Rhode Island College Providence. 02908
I
An undergraduate experiment using the Spectronic 20
Fluorescence spectroscopy is a topic which is usually neglected i n t h e undergraduate laboratory. The cause of this neglect is undoubtedly t h e absence of fluoredcence instrumentation in most chemistry departments. T h i s is unfortun a t e in light of t h e importance of fluorimetry as a sensitive analytical and research tool. Recently in this Journnl, Duncen, Kirkpatrick, and Neas described a simple method of modifying a Bausch and L o m b Spectonic 20 to a filter fluorometer ( I ) . T h i s development affords most d e p a r t m e n t s the opport u n i t y of offering challenging fluorescence experiments in undergraduate laboratory courses. T h i s paper describes an interesting experiment which utilizes fluorescence intensity measurements to determine t h e r a t e constant for t h e fluorescence quenching of various aromatic hydrocarbons b y CCb in an ethanol solvent. T h e experiment has been successfully performed in o u r physical chemistry a n d self-paced general chemistry courses.
Theory Aromatic hydrocarbons generally show intense fluorescence but no phosphorescence in fluid solution a t room temperature. The fluorescence of most of these compounds is efficiently quenched by halogenated alkanes such as carbon tetrachloride and chloroform (2-5). To a certain extent these quenehing events result in photochemical reactions between the aromatic hydrocarbon and the quencher. The object of this experiment is to determine the rate constant for this quenching reaction through measurements of the fluorescence intensity of the aromatic hydrocarbon in ethanol with various amounts of the quencher. The theory will he discussed with reference to the quenching of anthracene fluorescence with CCb in ethanol. We have also used perylene as the fluorescent molecule. The method employs the Stern-Volmer mechanism (6, 7) and is easily understood by undergraduate students. When ultraviolet lightisshone on a solution of anthracene inetha n d , a given anthracene molecule may absorb the light and in the process get converted to one of its excited singlet states. If astate other than the lowest excited singlet state becomes populated, the molecule will very rapidly (-10-L2-10-~J s) relax to the lowest excited singlet state. The anthraeene molecule in this state is denoted by A* and can now do one of several things. I t can emit a photon and in the process get converted back to the ground state. This is fluorescence and the fluorewence decay process is a first-order rate process with a rate constant denoted by kf.
divided by the rate at which it decays via both fluorescence and nonradiative decay.
If CCb is present in the solution, a third mechanism for the excited anthracene molecules to become depleted is available. This is the quenehing of the anthracene by the CCl+and is assumed to be second-order with rate constant k,. A.
+ CCb
-
Quenching
This quenching process occurs through several different paths. I t may result in ground state anthraeene and heat or in triplet state anthracene and heat. In the latter case the triplet state anthracene will usuallvrelax back to the eronnd state with therelease ofheat. I t is also possible far the triplet&thracene to react with CCb to a certain extent. In addition, the quenching of the excited singlet state will occur via a photochemical path to a certain extent.' It has been found that there are about eight different products for this reaction. Two of the products have been identified as 9-chloro- and 8,lO-dichloroanthraeene. The chief photochemical product, however, appears to be 9-chloro, 10-trichlaro-methyl,9,lO-dihydroanthracene.All of these quenehing processes of anthracene fluorescence by CCll are lumped toeether in the second-order rate constant k.. The net effect of the presence of CCI, is a reduction in the quantum yreld of fluures~enee of anthracene to the folloxing value!
m=
ki IA*l kl [A*] k,, [A*] k, [A*] [CCld
+
+
-- k f + k,, +kik,
[CCbJ
By dividing m0 by 9 we get the relative quantum yield. This is the Stern-Volmer expression 9019 = l
+kq [CCb] kt + k.,
(3)
The relntivequanlum yleld in this expressran can be replaced by the relatiw fluorescenre intensity I ' I which rs easily rneawred. A plut ul 1 I versus [CCI,] should yield a s t r a l ~ h lmt. t with n s h p e hq iki + k",). In order to determine the quenching rate constant k,, the (kr k,,,) must be determined by an independent experiment. It will be shown below that the measurement of the time dewndenee of the anthracene fluweacmcederay in pure ethnnalran be "red toohtain this. In the abjcnce uf CCI, the rate of disappearance of excited anthrarene is given by
+
A*-A+hu A second possibility is for the excited anthracene molecule to lose its energy in the form of heat rather than light. In this nonradiative process, the anthracene either gets converted hack to ground state (internal conversion) or to the lowest triplet state and then to the ground state (intemystem crossing). Noemission from the triplet state (phosphorescence) has been observed far anthracene in ethanol a t room temperature. Bath routes result in the disappearance of anthracene in its excited singlet state without the emission of light and can be lumped together in a first-order rate process with rate constant denoted by k,,. A' -A
+ heat
The only other first-order process would be a photochemical reaction hut this d w s not occur for anthracene in pure ethanol to a significant extent. The quantum yield of fluorescence, 9O, for anthracene in pure ethanol is the rate at which excited anthracene decays via fluoreseence
Therefore
w= -(kr + k",) dt [A*]
Integrating between the limits [Ao*] and [At*] on the left and 0 and
t on the right we obtain lag [At*] = log [Ao*] -
(k + k ) t 2.303
(6)
Only photochemistry coming from the excited singlet state will cause fluorescence quenching. Photochemistry fmrn the triplet state can also occur but will not cause fluorescence quenching. V t should be mentioned that the derivation of eqn. (2) assumes that the presence of CC4 has no effect on the first-order rate constants ki and k,,. Volume 54. Number 3, March IS77 1 183
molecule to became quenched by CCll molecule a t a given concentration of CCla. The value obtained can be considered to be a lower limit on the time it takes for a given anthracene molecule to collide witb a CC4 molecule a t the concentration of CCll used in the calculation. At a concentration of 1.0 M CClr the average time it takes for a given excited anthracene molecule to become quenched by a CCll m&cule is equal t o the reciprocal of the quenehing constant or 3.6 x 10-lo s. Suggestlono for Further Work This experiment can be extended to other aromatics which fluoresee such as phenanthrene and naththaeene and to other halogenated alkanes such as chloroform, bromofarm, and hexaehloroethylene. In addition, the experiment can be extended toinclude the direct photochemical quantum yield which can be measured a t various concentrations of CC4. A chemical actinometer can be used t o get the number of photons absorbed. The number of maleculea of anthracene reacted can be determined from the change in theabsorbance in the near uv of the solution after ohotolvsis for a certain oeriod of time. The ~ r n d u c of t this reaction does not absorb in th; near uv. The - - ~ r~~~~~~ measured quantum yield may be compared witb the quantum yield of quenching obtained from the fluorescence quenching experiment. ~
quenehing =
Stern-Volmer plot for the fluorescence quenching of anthracene by CCI. in ethanol ~alutlon.The concentration of anlhracene was 4.0 X lOP M f w all soIuti0nJ.
Since the fluorescence intensity is proportional t o the excited anthracene concentration, the fluorescence intensity can replace the excited anthracene concentration in the above equation. The students are provided with fluorescence lifetime data simulated from data taken by Dr. Arthur Halpern a t Northeastern University using the single photon counting technique. These lifetime data for both anthraeene and perylene are presented in Table 1. The students plot the log of the intensity versus time and from the slope determine (kt k,,). Once (k, k,,) is determined, the value of the quenehing rate constant can be determined from the Stern-Volmer plot.
+
+
Procedure The anthracene concentrations used in this experiment depend on the wavelength of the exciting light. I t is important that the optical density of the solution is not more than about 0.230 that the fluorescence intensity is uniform throughout the solut~on.I t is also important that all sample tubes be filled to the same level. If366 nm light is used to excite the anthracene its concentration should be 4 X M. If perylene is used as the fluorescing molecule the same concentration should be used. The CC4 concentrations should range from 0 to about 0.20 M for the anthracene quenching and from s 1 . 0 M for the perylene quenching. I t is important t o keep these solutions out of bright light so that the fluorescence intensity measurements take place before any of the fluorescing molecule disappears through the photochemical reaction. If the spectronie 20 is used for the fluorimeter a 0-51 Corning filter may be used to prevent scattered exciting light from hitting the photo tube as described in reference (I). However, this is not absolutely necessary since the amount of scattered light is small and can be nullified by zeroing the instrument with a pure ethanol reference. Discussion of R e s u l b The Stern-Volmer plot for anthracene obtained usinga Spectronic 20 is shown in the figure. The slope, k,l(kf k,,), has avalue of 14.1 I mole-'. This value is in excellent agreement with results using a Baird Atomic Fluorospee Fluorometer and with the literstive value of 14.3 1mole-L. The fluorescence lifetime plot yielded avalue of ( k f + k..) ... . of 2.0 X loa ss-'. When this is multipled by the Stern Volmer slope, a value of 2.8 X log l mole-' s-l is obtained for the quenching constant, k,. The results for both antbracene and perylene are summarized in Table 2. It is interesting to compare the value of k , with the diffusion controlled rate constant kd of 6 X lo9 1mole-'s-' (8).One might conclude from this that rauehlv half of the collisions between excited anthracene and CClr resilt i n quenehing. The students are encouraged t o determine from k,, the average time it takes for an excited anthracene
k, [CCIdI
(11)
k, + k", + k , [CCl4I
This expression yields a value of 0.64 when the CClr concentration is 0.125 M. At the same CCb coneentration, we found the photochemical quantum yield to be 0.39. Since this value includes reaction occurring from the triplet state of anthracene as well as that from the singlet it can be considered to be an upper limit an the singlet photochemical quantum yield. Comparison of this with the quantum yield of quenching leads one to the conclusion that less than 40% of the quenching events results in photochemistry. The authura would like to express his appreciation to Dr. Arthur Halpern for taking the fluorescence lifetime data and to the Hhode 14and College Hesearch Committee for a grant.
Literature Cited Kirkpstriek.J. W.,and Nea8.R. E.. J. CHEM, EDUC., 49,550 119721. (21 Bowen. E. J.. Tmna. Foradmy S u r . 50.97 11954). 131 Meluish, W. H..and MeLcslf,X.S.,J. Chem Soe, 480119681. (4) Lei*, M. S. S. C.. Rarinagui. K.. Chsm. Phys Lett.. 1.35 119691. (11 Dunean,R. L..
(5) Ware, W. %and Lewir,C., J, Chcm. Phys.. 57,8546 119721. 161 Storn.O..and Volrner, M..Physik. 2..20.18311919). 17) Calvert,J.%and Pilu,J. N.,"Phatoch~mialry.llJohn Wiley & Suns,lnc., New Yurk. 1966. . DD. . 663-70. (81 Reference 17J.p~.626-7.
Table 1: Fluorescence Lifetime Data for Anthracene and
Time ("4
Pervlsns
Intensity lphotonr/unit time1 ~erylem
~nfhracene
+
184 1 J m l of Chemical Education
Table 2. Summary of Rssuitr Pervlene
Anthracene
Fyaction effective coillrionr Photochemical quantum yield of disappearance
14.1 I m o d 2.0 X lo"-' 2.8 x l o s I mole-' r-' 6 x 10' I mole-'s-'
mote-' lo's-' 1.5 x 10" mote-' r-' 6 X 10' I mole-' r-' 1.4 X
1.1 I
112 0.39 at CCI, = 0.125 M
0.16 at CCI,
= 0.82
M
