Fluorescence Quenchingof Acridinium Ions in Sodium Dodecyl Sulfate Micelles El-Zeiny M. Ebeid Tanta University. Tanta, Egypt When a molecule absorbs light, it is excited to a higher energy electronic state which exists for a short time before losing the excitation energy via one or more of the routes shown in Figure 1 ( 1 , Z ) . Since these routes of deactivation are competitive with fluorescence,they reduce the number of photons emitted and so cause fluorecence quenching. Fluorescence quenching may he caused by added quenchers that enhance one or more deactivative routes other than fluorescence, e.g., intersystem crossing (isc) or internal conversion (ic). Two types of fluorescence quenching are well known, namely collisional and static quenching (3). The present experiment demonstrates a collisional quenching of fluorescence. For an individual molecule in an excited state, there is a certain probability of decay in a given time interval. For a sample containing N molecules, the rate of decay is thus proportional to N , i.e.,
proportion to k - l , i.e., N .- k-'. On the other hand, the intensity of fluorescence, I, is proportional to N; it follows that I = k-', i.e., k =I0 ko I
Substituting in eqn. (2) for klko, we get
where lo is the intensity of fluorescence with no quencher
and N decays exponentially; k is the rate constant of decay, and the reciprocal of k gives the decay time. The term k is the sum of all rate constants for the various deactivation processes, and in the presence of a quencher of concentration [Q], we often find k = ko + kJQl
I.e.,
where k q is the second-order rate constant for quenching. In steady state experiments, the rate of decay equals the rate of excitation, which is usually fixed by the intensity of exciting light. In eqn. (I),then, d N l d t is fixed, S O Nvaries in
Figure 1. An illusbation of some rwtes of deactivationof an elechonically excited singlet state 1%). TI, first excited triplet state; SO,ground state: -, radib tionless deactivation. 1e.g.. (a), chemical reaction; (b), internal convenlon (Ic); (c) and Id), intersystem crossing (isc)); Radiative deactivation. (e.g., hv,, fluorescence: hu,, phosphorescence; --, light absorption).
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Volume 62 Number 2
February 1985
165
filter uf maximum trar~stniamnaround Rfi; nm, and, for more daborate work, a ~imyletluor~mewri*needed. Experimental Check, under the 365-nm light,the green emission of a 10WMAHf solution.The emission maximum is found to be 460 nm using a fluorimeter. To 1 mlof the 10-'MAHCsolutionadd 8ml ofdistilled waterand 1ml of NaBr solution, and observe the intensity of the green emission, which will he much weaker than that of the lOWM solution. If a fluorimeter is used, it will be of interest to compare solutions made up using 1,2,3 ml . . .of NaBr solution, all with 1ml of AHt and made up to 10 ml with distilled water. A graph may then be plotted of the intensity ratio (I& versus [Br-1. The gradient of this graph gives the value (k,lko). Since (llko) is the decay timer of AH+ in the absence of quencher, then to obtain k, in the usual units of dm3mol-' s-I we must divide the . aradient value. by- 7 (Tfor AHt is 32 X 10-gs) (5). To see the effect of mieelles make uo a solution bvmixine 1ml AHt. 8 ml SUS msrlle iolurm, and I rnl C s ~ r . T h i s t & ethe t~uoresrrnre appears unqurnrhrd tior an exact compari,on use 1 tnl AH', 0 ml SDS, and 1 ml water). The experiment can he repeated, substituting CoSOa solution for NaBr. In three separate test tubes mix the quantities shown below. ~
Figure 2. An idealized model for a sphwlcal ionic micelle. The circles represent ionic head groups.
~
~~
Emerirnent present and I is the intensity of tluorcscence in the presence of quencher of concentrnrion 191.A graph of ( l o l l ) versus [Ql is called a Stern-Vdmer plot and is often usrd to investigate the kinetics of the qunching process. In the present experiment, Br- or Co2+ are used as quenchers of acridinium ions (AH+) (4,5). H+ A ~ . A
&ggKJ
It also demonstrates the micellar effect in the area of surface chemistry. Micelles are groups of associated surfactant molecules. Surfactant molecules usually have an ionic or polar hydrophilic head group and long hydrophobic organic groups. Above a critical micelle concentration (cmc). thev associate as micelles in which the electrostatic repulsionsbetween the ionic heads are oonosed . bv hindine between the hvdro~hobicorganic groups as sho& in ~ i i u r 2. e ~ o w e v e ithis , model is dynamic, micelles being formed and disrupted in equilibrium with monomeric surfactant in the medium (6, 7). I n sodium dodecyl sulfate (SDS) surfactant, the polar group is the -0S0; group and the hydrocarbon group is -C12H25
.
Materials Dissolve 10-4 mmol of acridine (caution:8 skin irritant) in 11of M aqueous sulfuric acid. Such acidity ensures protonation af most of the acridine giving acridinium ion, AHf. The solution thus obtained is stable over lone -.oeriods of time bears when stoooered). .. Solutions of l O V M NaBr and 10-4 M CoSO4 are prepared in distilled water (NaI can replace NaBr in all experiments). Sodium dndecyl sulfate micelle is prepared by dissolving -2.9 g of sodium dndecyl sulfate (SDS) (also known as sodium lauryl sulfate, ClzH2sOSOaNa) in 50 ml of distilled water. Since SDS is an ester, it undergoes slow hydrolysis in aqueous solution, and it is recommended that a stock solution should not be stored for more than a month. Fluorescence is produced using a UV lamp combined with a glass ~~
~~~
ml H"0
ml CaSO.
ml SDS
Fluorescence intensity in Experiment (1)is taken as a reference, and in (2) and (3) fluorescence quenching will be observed. Discussion Fluorescence quenching hy Br- or I- is an example of the heavy atom effect (2) whereas fluorescence quenching by Co2+ is due to electronic energy transfer from the electronically excited acridinium ion to Co2+ ions via a Fbrster-type mechanism (2,8). In micellar solutions, the negative polar heads are subjected to interactions with positive ions, e.g., AH+ and Co2+whereas negatively charged ions, e.g., Br- or I- are repelled. Fluorescence quenching of AH+ is thus caused by Co2+ in micellar solutions as they come close together by co-adsorption on the micelle's surface whereas no fluorescence quenching is caused by Br- or I- for AH+ in micellar solutions because a physical separation of the fluorescer (AH+) and the quencher (Bror I-) is introduced by the negatively charged micelle's surface. Acknowledgment
I thank N. J. Bridge for useful discussions Literature Cited
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166
rnl AH'
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Journal of Chemical Education
11) Caivert, J. G.. and Pittr, J. N., "Photachemistry," John Wiley and Sons, Nev York, ,an7
12) Cunddi, R B., and Gilbert, A,, "Photochemistry," Nelson,London, 1970. 13) Penzer, G.R.,in "An Intraduction to Spectrascopy for Biochemistr,"(Editor: Bmm, S.B.), Academic Press, 1980, p. 83. (4) Keh, E., and Valeur, B., J. Call. Interlace S c i , 79,465 (1981). 15) Bridge, N. J., and Fletcher, P. D. I.. J. Chom Soc. Faradqv TIOM. I , i n p r w . 16) MeC.aldin,J. 0.. and Somorjsi. G..Plog Solid Stofe Chem., 8,239, (19731. 17) Topics Current Chom.. 87. (1880).s special isaue about miedln. IS) Kemlo,J.A.,andShepherd,T. M.,Ckrn.Phya. Lett.,47,158(1977).
