Glles Henderson Eastern Illinois University Charleston. 61920
I The Effects of Absorption and SelfI Absorption Quenching on Fluorescent
I Intensities
Instrumental analysis students are frequently exposed 1,) the cuncept that fluorescence intensities are proportional to the nower of the excitation beam that is absorbed by ..- - radiant -- - the sample (1-5).Although this is indeed true, acareful consideration of the optical system and self absorption, sometimes called the "inner filter effect" (6),must he included to properlv, understand the effect of sample concentration on emission intensities, particularly in fluorescence excitation spectra. Most instrumental texts (I-.5) and even standard fluorescence monographs (7-11) do not really present absorption effects very clearly, if a t all. In general, excitation spectra exhibit apparent concentration-dependent wavelength shifts (see Fig. 1). The naive student might well interpret this phenomenon ar a conrentration.depe~dentperturbation oft he molecular enerm Ie\ds. However, this postulate is clearly ruled out since absorption spectra of solutions of the same concentrations obey Beer's law and do not exhibit such wavelength shifts (see Fig. 2). The actual cause of this phenomenon will be developed in detail in this paper. ~
~
~~
Effect of Optical Parameters and Concentration on Fluorescence 'i'he approach used here to derive the observed fluorescence incensitv is similar to that used hy Winefordner and wworkers 172 ~ - -73i , hut simnlified to facilitate discussion of basic concepts. The optics employed in a typical spectrofluorometer do not interrogate the total cross-section of the sample cell. Excitation radiation is focused into a beam whose width is determined in Dart bv the slit widths of the excitation monochrumator (see Fig. 3). likewise the emission monorhromiltor "sees" only a portion of the fluorescent radiation heam. Thus if we neglect scat~eredradiation, the instr~lmentmeasures onlv the fluorescence produccd within the intersrctiny: regions of the excitatwn and emission bcamg. The tluoreicent inrena l the radiant power of the excitation sitv L' is ~ r o ~ n r t i o nto beam that iiabsorhed by the samplein this region
. ~ ~ ~ - ~
~
~~~
F = $,(Po' - P )
(1)
where Po' is the power of the excitation beam after traveling a distance lI through this sample (see Fig. 3), P is its power after traveling a length l1 12, and 4, is the fluorescent quantum yield, a constant which depends on the efficiency
+
WAVELENGTH (nm) ergwe 1, & mentation of biacetyl in CCI,.
dependence of
fluaescence
$wrnFigure 2. The concentration dependence of the absorption specburn of biacety
exci~im
in CCI,.
Volume 54, Number 1. January 1977 / 57
1 I Figure 3. The optical parametars ofthe samle cell for atypical spctrophotofluommeter.
0
.iZ .I8 MOLARITY
.06
30
.24
Figure 4. The concentration dependence of observed fluaescence intensities. I = ,422 The circled points represent experimentalmeasurementsfa. A, = , nm and )g, = 464 nm. The solid curve corresponds to the relative intensity profile calculated from eqn. (6).
of the fluorescence process. Using Beer's law where 6,is the molar absorptivity of the fluorescent molecules a t the excitation wavelength X.;, then
F = 4,por(l - e - * d ~ c )
(3)
It is important to recognize that both the excitation beam and the emission undergo absorption in regions 11 and 13, respectively. In fact it will be shown that it is indeed these absorption processes that result in the apparent concentration dependent wavelength shifts of the excitation spectra (Fig. 1). From Beer's law we have where Pois the power of the excitation beam incident upon the sample. Substituting eqn. (4) for Po'in eqn. (3) gives F = p 04re - * d ~ c ( l- e - e e d w )
(5)
Finally, the intensity of fluorescent emission is diminished by self absorption over the distance 13. Again from Beer's law we have where I is the observed fluorescence intensity, k is an instrumental constant to correct for the fact that the fluorescence is emitted isotropically but is observed only through a small aperture, and c,. is the molar absorptivity of the fluorescent molecule a t the emission wavelength he,. Experimental All of the data presented here were obtained with solutions of 2,3-hutanedione (biacetyl) in reagent grade CCl,. T h e hiacetyl (Eastman Kodak 1591) was used without purification. Excitation soectra and fluorescence intensities were measured with an Aminco-Bowman spectrophotofluorometer. Fluorescence emission was measured a t 464 nm using a slit width of 0.05 cm for both the excitation and emission monochromators. The excitation svectra are uncorrected; i.e. they are n o t n , r r c < ~ r for d the wa&length dependtmcc of the phutumulti~lier'swnsiti\.it\., the transmi;sion of the monnchn,. mato;, or the hand width of the monochromator. The fluorescence measurements were made with a l-cm quartz cuvet (1.1 = 11.5rnll.Since both monochromators ha\.e idcntiral uptir;il deiirn. I, can he measured by placing n piere of film in the sample c&et normal to the excitation beam and irradiating it for a brief moment. For our instrument with a 0.05 cm slit width, l z = 0.196 cm, and 11 = 13 - 0.512 = 0.402 cm. Absorption spectra were measured with a Beckman-Acta model M-IV spectrophotometer. Molar ahsorptivities were calculated from Beer's law 58 / Journal of Chemical Education
Figure 5. Fluorescence intensity profiles and their components in regions of Strong absorption (upper plot). moderate absorption (middle plot), and weak absorption (lower plot). The - - - curve represents the relative fluorescence incurve corresponds tensities produced in the region h ofthe cell; the upper to the self absorption of emission energy in the region h; the lower - curve represents absorption of excitation energy in the region I, and the solid curve represents the overall relative fluorescence intensity, the product of the above three components. See eqn. (8).
- .-
-.
Results and Discussion The molar ahsorptivities
