Reduced Fluorescence from Tris-(8-quinolinolato)
Aluminum(III) in the Presence of Its Fe(III) Analog
P A U L J. MILHAM,* ALUN W. HUDSON, M I C H A E L J. MAGUIRE, K A M E L S. HADDAD,
a n d C O L I N C . SHORT
New South Wales Department of Agriculture, Biological and Chemical Research Institute, P.M.B. 10, Rydalmere, 2116,
Australia
Chloroform (CHCh) s o l u t i o n s o f tris(8-quinolinolato) Al(III)
(AIQ3), w h e n e x c i t e d at 395 nm, f l u o r e s c e at 520 nm. A d d i t i o n of
tris(8-quinolinolato) Fe(III) (FeQ~) to t h e s e s o l u t i o n s d e c r e a s e s
the fluorescence: the ratio of the f l u o r e s c e n t i n t e n s i t y in the
p r e s e n c e o f FeQ3, to that in its absence, is e x p o n e n t i a l l y related
to [FeQ3] and i n d e p e n d e n t of [A1Q~].We h y p o t h e s i z e d that the
main c a u s e o f this effect w a s FeQa a b s o r p t i o n o f the e x c i t i n g
and e m i t t e d radiations. The h y p o t h e s i s w a s t e s t e d in t w o w a y s :
first, by c a l c u l a t i n g f l u o r e s c e n c e l o s s e s u s i n g FeQ~ a b s o r b a n c e
m e a s u r e d at 395 and 520 nm; and second, b y m e a s u r i n g fluores-
cence l o s s e s w h e n the AIQ3 w a s in one optical cell, and the FeQ3
in others, w h i c h w e r e l o c a t e d in either the e x c i t a t i o n or e m i s s i o n
paths, or both. C o m p a r i s o n b e t w e e n all the r e s u l t s s h o w e d that
t h e y w e r e n o t s i g n i f i c a n t l y different (Po.o0, t h u s v e r i f y i n g the
hypothesis.
Index Headings:F l u o r e s c e n c e o f tris-(8-quinolinolato) Al(III);
Tris-(8-quinolinolato), Fe(III) inner filter effects on; Trace de-
t e r m i n a t i o n o f A1.
INTRODUCTION
Al(III) and Fe(III) ions in aqueous solution react with
8-quinolinol (HQ) to form tris complexes (A1Q~and FeQ3,
respectively) which are readily partitioned into CHC13.1' 2
The absorbance spectra of the extracted complexes over-
lap, interfering with quantitation of A1Q~.1' 2 This inter-
ference has been reduced by prior separation or masking
of the Fe, 2-5 or by taking advantage of the intense green
fluorescence emitted by A1Q~ (~.... - 520 nm) when it is
excited at -395 nm. 4-~ Although other techniques have
been developed for determining trace concentrations of
A1, methods based on the fluorescence of A1Q~ continue
to be used for the analysis of soil extracts 7' s because they
are sensitive, discriminate between mono- and polynu-
clear A1 cations, 9' lo and are reported to be relatively free
from Fe interference.GA1 concentrations determined in
this way have been used to derive relationships with soil
pH and with plant growthJ' 11.12
No detailed work has been published on the effect of
FeQ~ on the fluorescence of A1Q~; however, the phenom-
enon has been noted. 4-6 Noll and Stefanelli4 reported no
fluorescence loss when solutions were spiked with 5 #g
F e m l -~, but the conditions of the experiment were poorly
defined. Goon et a l J and Cook~ both used similar exper-
imental conditions and design (one [All and several of
Fe), and although each used a different [All, they inde-
pendently reported fluorescence reductions of -40% with
1.2 ~g F e m l -~ in the CHC13 extract of the complexes. The
agreement between results was quantitatively poorer at
Received 29 November 1978.
* Author to whomcorrespondenceshouldbe addressed.
298 Volume 33, Number 3, 1979
0.8 #g F e m l -1 CHC13, the only other concentration at
which data are available for comparison. Because Cook
observed a large fluorescence loss when Fe/A1 (w/w) >
1, he assumed that at other A1 concentrations, substantial
interference would occur if, and only if, Fe/A1 (w/w) >
1. Although this assumption is contrary to the data of
Goon et al., it has gone unchallenged.
We present data for a number of Fe and Al concentra-
tions in factorial combinations which show that FeQ3
reduces the fluorescence from A1Q3 as a function of [Fe]
and not of Fe/A1. Complementary spectrophotometric
and fluorimetric techniques are used to elucidate the
mechanism of the effect.
I. EXPERIMENTAL
Aqueous solutions containing A1 at 0, 2, 5, and 10 ~g
m1-1 and Fe at 0, 5, 10, 20, 50, and 100 ~g m1-1 were
prepared in factorial combination (i.e., 24 unique combi-
nations) with two replicates. An aliquot (2.5 ml) of each
solution was diluted with distilled water (50 ml), excess
8-quinolinol was added, the pH was adjusted, and the
complexes were extracted into CHCI~ (25 ml). ~ The
CHC13 extracts were dried over sodium sulfate.
Fluorescence data were collected using a Farrand MKI
fluorimeter (Farrand Optical Co. Inc., New York). The
cell compartment was modified to locate up to three
optical cells in the light path simultaneously and to allow
masking options on one cell (Fig. 1). The UV-visible
absorption spectra were recorded (380 to 550 nm) using
a Varian-Techtron model 635 D spectrophotometer fitted
with 5 mm path cells (Varian-Techtron, Melbourne, Aus-
tralia).
II. RESULTS AND DISCUSSION
A. Fluorescence Suppression b y F e Q 3 . Irradiation
of CHC13 solutions of A1Q3 at ~395 nm caused fluores-
cence at ~520 nm, as reported previously. 13 The fluores-
cent intensity was almost a linear function of [A1Q~].
Under the same excitation conditions, no fluorescence
was observed (400 to 700 nm) from FeQ3. In solutions
containing both complexes, A1Q3 retained its character-
istic maximum excitation and fluorescence wavelengths
and its fluorescence band half-width; however, the inten-
sity of the fluorescence was reduced.
A plot of fluorescent intensity vs [Fe] (#g m1-1 CHC13)
yielded a family of curves, one for each [All. If, instead,
the fluorescent intensities (Fi), at constant [All, were
expressed as a proportion of the intensity in the absence
of Fe (Fo), plots of the relative fluorescent intensity ( F i /
Fo) vs [Fe] were coincident for all [All (Fig. 2). This
showed that Fi/Fo was independent of [All. We therefore
APPLIED SPECTROSCOPY
 difference appears to have been caused by the approxi-
mately 3 times greater length of the radiation paths
through the optical cells of the fluorimeter. The signifi-
cance of the path differences between fluorimeters will
011 I become obvious later. More importantly, our data con-
flict with Cook's assumption that Fe reduces fluorescent
intensity if, and only if, Fe/A1 (w/w) > 1. However,
inspection of Cook's experimental design shows that it
I I was inadequate to distinguish between Fe/A1 and [Fe]
c b al
M~--I--I--I- I effects.
To throw some light on the mechanism of the FeQ.~
effect, we measured fluorescent intensities under condi-
h- - , - - ; - [ 'T C 2 - tions which allowed us to vary independently the dis-
M2 C3 tance traveled by the exciting and emitted radiations
FIe. l. Schematic diagram of horizontal section through the modified through the CHCI~ solutions. This was achieved by using
cell compartment of the fluorimeter. The locations of the three cells the nine masking combinations, ala2, ... clc2 (with masks
(C,, C2, and C3) and the two masks (M, and M2) on the central cell are 1 and 2, respectively, Fig. 1). Under these conditions, and
indicated. Each mask had a single 1 mm diameter aperture which was at all of the FeQ3 concentrations tested, fluorescence was
either centered in the face of the window (location b) or displaced
horizontally by 3 mm from it (locations a and c).
increasingly inhibited in the order: alaz < a~b2 ~ blaz <
a,c2 = bib2 ~ cla2 < blC2 ~ clb2 < CLC2. The sums of the
I I I
respective pathlengths of the exciting and emitted radia-
1OO tions of these five groups were approximately 4, 7, 10, 13,
>,
and 16 mm. Significantly, the relative proportions be-
tween these distances were almost identical to those
E between the corresponding (logarithmic) fluorescence in-
E hibitions. Hence we inferred that FeQz reduces fluores-
o

03 50 cence by partially absorbing both the exciting and emit-
O ted radiations, the absorptions being similar in magni-
tude per unit length of either path and linearly propor-
tional to the pathlength.
B. F l u o r e s c e n c e Loss C a u s e d b y t h e FeQ~ F i l t e r
13 Effect. We decided to test the proposed filter mechanism
I ~ I , I by comparing calculated fluorescence losses with those
0 5 10 measured using the unmasked cell [Eq. (3)]. The fluores-
cence loss was calculated (Section II B3) from the esti-
jug Fe mL-1 CHCI3
mates of the mean pathlengths of the exciting and emit-
Fro. 2. Relative fluorescent intensity at three different A1 concentra- ted radiations (Section II B1) and measurements of FeQ,
tions [0.2 (), 0.5 (ff]), and 1.0 (A) ttg ml-' CHC13] as a function of Fe absorbance for a known length of each path (Section II
concentration.
B2).
1. E s t i m a t i o n o f M e a n R a d i a t i o n Pathlengths. Obser-
bulked the relative intensity data for all [All to derive vations of fluorescence in the absence of FeQ3, with
the general relationship masking locations az, b2, and c2 and without mask 1 (Fig.
logt0 (Fi/Fo) = -0.081 [Fe] (1) 1), showed that the fluorimeter was at least 10 times
more sensitive to fluorescent radiation which originated
The fluorescent intensity loss (L) can be expressed as within 0.5 mm of the optical axis of the fluorescence
L = (log,o Fo-log,oFi) monochromator (location bz), than to that which origi-
nated 3 0.5 mm to either side of the axis (locations a2
= log~o(Fo/Fi) (2) and c2). This suggested that if the fluorescence cell were
= -logio(Fi/Fo) unmasked, the major proportion of the detected fluores-
cence would have originated near the optical axis of the
so from Eq. (1) fluorescence monochromator (mask location b2, Fig. 1);
L = 0.081 [Fe] (3) and that to reach this axis the exciting radiation would
have traveled a mean distance of ~5 mm through the
for which the square of the coefficient of correlation (r e) CHCi.~. Commencing with this value for the mean ab-
was >0.99. An identical equation was also derived from sorption path of the exciting radiation, we used two
fluorescent intensity data (not presented here) which independent procedures to estimate the mean distance
were obtained by serially diluting CHCI~ solutions of the of travel of the emitted radiation through the solution.
complexes. First, we compared the fluorescent intensities for the
These results are qualitatively similar to those re- unmasked configuration and for the five masked groups
ported by Goon et al. 5 and Cook,6 but they are quanti- between which the inhibitions differed. This revealed a
tatively different. For example, we found only about one- close similarity in magnitude only for the 10 mm path
third of the ~40% loss of fluorescence which they both group, i.e., a~c2, bib2, and clae (Fig. 1). Of these only the
observed at 1.2 /tg F e m l - ' CHCI~. In one case, 5 this bib2 combination has the required 5 mm mean absorption

APPLIED SPECTROSCOPY 299

path for the exciting radiation, which leads to an estimate
of 5 mm for the mean absorption path of the emitted
radiation.
Secondly, we used a photographic technique to meas-
ure the intensity distribution of the exciting radiation
intercepting the vertical plane through the optical axis of
the fluorescence monochromator. By regarding the cen-
ter of gravity of the distribution as the origin of all the
fluorescence occurring in the plane, we again arrived at
a mean absorption pathlength of 5 mm for the emitted
radiation.
2. Characteristics of Energy Absorption at 395 and
520 nm. The absorption spectra of FeQ3 and A1Q~ had
~maxvalues near 465 and 395 nm, respectively. The tails
of the FeQ3 band spread through 395 and 520 nm, the
respective wavelengths used to excite and measure emis-
sion from A1Q~. However, AIQz did not absorb signifi-
cantly at 520 nm.
At each wavelength, the absorbances of the complexes
were independent and followed Beer's Law. Therefore,
FeQ3 absorbance values at 395 and 520 nm have been
averaged for all [All and are presented for 5.0 mm paths,
in keeping with the estimated lengths of the mean exci-
tation and emission paths [Eqs. (4) and (5)].
A395 = 0.047 [Fe] (4)
Aseo = 0.033 [Fe] (5)
In each case, r e > 0.99.
3. Fluorescence Loss Calculation The total fluores-
cence loss, L [Eq. (2)], can be regarded as the sum of two
(logarithmic) components, 1395 and 15e9, i.e., the losses
occurring at 395 and 520 nm, respectively.
Consider the quantitative relationship between 139~and
A39~. Fluorescent intensity is proportional to the intensity
of the exciting radiation and the proportionality constant
is the quantum yieldJ 4 If the quantum yield of A1Q~ is
unaffected in the presence of FeQ3, it can be shown that
the numerical values of 139~and A395 will be equal, i.e.,
from Eq. (4)
1395= 0.047 [Fe] (6)
Similarly it can be shown from Eq. (5) that
1520= 0.033 [Fe] (7)
Because
L =/395 -t- 152o (8)
we can write, using Eqs. (6) and (7),
L = (0.047 + 0.033) [Fe] (9)
i.e.
L = 0.080 [Fe] (10)
300 Volume 33, Number 3, 1979
Statistical comparison between the measured [Eq. (3)]
and calculated [Eq. (10)] values of L shows that they are
not significantly different (Po.ol).
C. D i r e c t M e a s u r e m e n t of/395,/520 a n d L w i t h FeQ3
a n d AIQ3 in S e p a r a t e Solutions. To test the filter
model in greater detail, we measured the fluorescence
with AIQ3 solutions located in cell 2 (Fig. 1) and FeQ3
solutions in cell 1 [Eq. (11)] or cell 3 [Eq. (12)] or in cells
1 and 3 [Eq. (13)]; i.e., in a system where the only possible
interaction between the complexes was an optical one.
The results were:
139~= 0.047 [Fe] (11)
(12)
1520= 0.033 [Fe]
L = 0.080 [Fe] (13)
In all cases r 2 > 0.99.
Thus, there is excellent agreement between the values
observed for L, whether [Eq. (13)] or not [Eq. (3)] the
complexes were isolated from one another. Moreover, the
observed L values are not significantly different (P0.01)
from that calculated assuming only filter effects [Eq.
(10)]. There is also excellent agreement between the
measured values for 1~9~and 152o,and the corresponding
values predicted from absorbance data [Eqs. (6) and (7),
respectively]. In no case was a statistically significant
difference found (P0.ol). Thus, the filter hypothesis ade-
quately accommodates all the observations.
Finally, it is worth noting that the similar magnitude
of la95 and 152oexplains the pathlength effects found in
our masking experiment. Likewise, the linear relationship
between L and pathlength explains why Goon et al., ~
with a light path ~3 times longer than ours, obtained an
~3 times greater fluorescence inhibition than we did, for
the same [Fe].
ACKNOWLEDGMENT
The authors thank Miss Wendy Morrison for preparing the figures.
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