Supporting Materials
Efficiency of Electron Transfer Initiated Chemiluminescence
Felipe A. Augusto, Glalci A. de Souza, Sergio P. de Souza Júnior, Muhammad
Khalid, Wilhelm J. Baader*
Departamento de Química Fundamental, Instituto de Química, Universidade de São
Paulo, São Paulo, Brazil
*Corresponding author: wjbaader@iq.usp.br (Wilhelm J. Baader)
1
Table S1: Quantum yields (E mol-1) for the decomposition of additional examples of
1,2-dioxetane
derivatives
containing
aromatic
hydroxyl
groups
initiated
by
deprotonation.
Structure
CL
S
t1/2 (s, 25ºC)
Ref.
1.4 10-1
-
75
(1)
4.4 10-1
6.3 10-1
≈11000
(2)
9.2 10-3
-
1.9
(3)
4.6 10-1
7.5 10-1
578
(4)
3.9 10-1
5.9 10-1
≈1600
(4)
1.8 10-2
-
13.9
(5)
2.0 10-2
2.0 10-1
41
(6)
3.7 10-1
-
44
(7)
1.7 10-1
-
10.0
(8)
1.9 10-1
-
147
(9)
2
In
all
cases
calibration
2.1 10-1
-
27
(9)
4.4 10-2
7.3 10-1
96
(10)
1.8 10-1
-
7.7
(11)
4.1 10-1
8.0 10-1
≈1500
(12)
relative
to
3-adamantylidene-4-(3-tert-butyl-
dimethylsiloxyphenyl)-4-methoxy-1,2-dioxetane.(13)
3
Table S2: Quantum yields (E mol-1) for the decomposition of some additional examples
of 1,2-dioxetane derivatives containing silyl-protected aromatic hydroxyl groups,
induced by fluoride deprotection.
Structure
CL
S
t1/2 (s, 25ºC)
Cal.a
Ref.
2.6 10-6
-
1.4
II
(14)
3.0 10-1
-
0.4
-
(15)
7.0 10-3
-
0.9
-
(16)
5.0 10-2
-
2.7
-
(17)
2.1 10-3
-
2.4
VI
(18)
2.1 10-3
-
22
VI
(18)
1.3 10-2
-
0.6
VI
(19)
2.5 10-1
7.6 10-1
9.8
VI
(20)
4
2.4 10-1
7.5 10-1
16.0
VI
(21)
1.5 10-2
6.3 10-2
178
-
(22)
TBS: tert-butyldimethylsilyl.
a
Calibration method – II: according to Hastings-Weber procedure,(23) VI: relative to 3-
adamantylidene-4-(3-tert-butyl-dimethylsiloxyphenyl)-4-methoxy-1,2-dioxetane.(13)
5
Detailed procedures for determination of chemiluminescence quantum yields
(i)
Calibration according to the Hastings-Weber Procedure (23)
Hastings and Weber established a method for chemiluminescence quantum yield
determination which utilizes a source of homogeneous radiation as calibration standard.
This secondary light emission standard consists of a radioactive toluene solution
containing hexadecane labeled in the 1-position with
14
C or tritiated hexadecane and
2,5-diphenyloxazole and 2,2’-p-phenylbis(5-phenyloxazole) as scintillators. The particles emitted by the
14
C or tritium can excite the scintillators either directly or
mediated by the solvent and the light emission obtained is identical to that observed
after photoexcitation of 2,2’-p-phenylbis(5-phenyloxazole).
The light flux of the Hastings-Weber tritium standard, prepared as described
above, has been determined to possess a light flux of IHW = 4.91 108 quanta s-1 cm-3,
using a complex instrumental set-up and methodology described in the literature.(23)
This standard, if available in the laboratory, can now be utilized for the calibration of
the emission intensity in chemiluminescence reactions, using the same geometric set up
for calibration and the reaction, thereby avoiding mathematically difficult geometry
calculations.
The emission intensity obtained in a CL reaction (IS), corresponding to the light
emission intensity of the sample in Einstein per second per liter [E s-1 L-1], can be
obtained from the sample emission intensity measured in arbitrary units (SS) by
comparing this intensity with the signal intensity of the Hastings Weber standard (SHW),
measured in exactly the same geometric and instrumental conditions (Equation 1). In
order to transform the units for the total light flux from [quanta s-1 cm-3] to [E s-1 L-1],
the former value has to be divided by Avogadro constant and multiplied by 103 to
6
transform cm3 to L. Additionally, the emission intensity has to be corrected for the
relative spectral sensitivity of the photomultiplier tube (PMT) utilized, using the ratio of
the sensitivity at the emission wavelength (max = 416 nm) of the HW standard (f(416))
and the emission maximum of the sample (f(S)); these relative values can be obtained
from the PMT fabricants. In this way the emission intensity of a given CL reaction can
be obtained in Einstein units which can be directly compared to the concentration of the
limiting reagent utilized in the reaction (Equation 1).
Equation 1
For a unimolecular reaction, the quantum yield for the transformation can be
directly determined from the initial emission intensity (in [E s-1 L-1]) and the
unimolecular rate constant (k, in s-1) of the transformation, utilizing the initial
concentration of the limiting reagent (Equation 2).
Equation 2
For more complex transformations, the quantum yields can be more
conveniently obtained by the integration of the emission intensity curves, determining
the total amount of light emitted by the reaction (AS), initially measured in arbitrary
units. These values can be transformed in Einstein units using the HW calibration
standard (ACL), in analogy to the transformation of light emission intensities (Equation
3).
Equation 3
It should be observed that the value used for the HW standard (IHW and SHW) are
the same here as in the intensity calibration, however, it is important to utilize the same
time units (seconds) in the integration of the light emission intensity of the sample. The
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emission quantum yields (in E mol-1) for the CL transformation can be obtained by
dividing ACL (in E L-1) by the concentration of the limiting reagent (in mol L-1).
(ii)
Calibration with the modified luminol standard (24,25)
A variation of the initially proposed methodology of the luminol calibration standard
has been utilized by our research group to determine quantum yields for a variety of CL
systems, including the peroxyoxalate reaction, the catalyzed decomposition of 1,2dioxetanones and the induced decomposition of 1,2-dioxetanes. (26) This method has
the advantage to be relatively easy to be performed and, most importantly in
comparison to the Hastings-Weber calibration, uses readily available reagents and
instrumentation.
In order to determine the CL quantum yields of complex systems, like the
peroxyoxalate reaction, the total light emission (Q) obtained in a kinetic assay is
initially determined in arbitrary units by integration of emission intensity versus time
curves. The conversion of the total light emission in arbitrary units to Einstein units (E)
is now performed by using the calibration factor (flum) obtained by measuring the
emission kinetics of the luminol reaction performed in standard conditions (see below)
and exactly the same instrumental and geometric set-up as the specific CL reaction. The
calibration factor is calculated from the integral of total light emission in the luminol
assay (Qlum), the luminol chemiluminescence quantum yield (lum = 0.0114 ± 0.0006 E
mol-1) and the number of moles of luminol (nlum) utilized in the standard assay
(Equation 4).
Equation 4
The chemiluminescence quantum yield for a given reaction can be obtained by
multiplication of the area under the curve of emission intensity versus time for a certain
CL reaction (Q, in a.u.) with the luminol calibration factor (flum) and the photomultiplier
8
sensibility factor (fphoto), divided by the number of moles of the limiting reactant (n) of
the CL reaction (Equation 5). The photomultiplier sensibility factor is obtained from the
PMT sensitivity at the emission wavelength (max = 431 nm) of the luminol standard
(f(lum)) and the emission maximum of the specific CL reaction (f(S)) (Equation 6).
Equation 5
Equation 6
Typical Experimental Procedure:
Stock solutions:
a) sodium phosphate buffer (0.1 mol L-1) pH = 11.6;
b) luminol ( = 7600 L mol-1 cm-1) stock solution in sodium phosphate buffer, in
an adequate concentration depending on the sensibility of the instrument to be
calibrated (10-9 to 10-3 mol L-1);
c) hydrogen peroxide (0.3% in water);
d) hemin (2.5 mg) in 10 mL of NaOH 1 mol L-1, diluted in NaOH 1 mol L-1 to two
new stock solutions with absorbance of 0.6 and 0.2 at  = 414 nm.
Luminol Calibration Procedure: A 3.0 mL quartz cell containing 2.8 mL of the luminol
stock solution is placed in the detection instrument and data acquisition is initiated,
followed by addition of 100 L of the hydrogen peroxide (0.3%) stock solution and 100
L of the diluted hemin solution (absorbance 0.2), when an intense CL emission is
observed. After the emission intensity approaches background level, 100 L of the
concentrated hemin solution (absorbance 0.6) are added and the procedure is repeated
until no more considerable light emission can be observed upon hemin addition;
normally the second or third addition of the concentrated hemin solution only leads to
9
very low light emission. The whole experiment is repeated at least five times in order to
obtain reliable results.
The experimentally obtained emission intensity versus time curves for all
successive hemin additions are integrated numerically, obtaining the total amount of
light emitted by the standard luminol reaction (Qlum) and this value is used to calculate
the calibration factor (flum) (Equation 4).
The luminol calibration procedure can be utilized in a wide range of luminol
concentrations (10-9 to 2 10-3 mol L-1), as the luminol chemiluminescence quantum yield
remains constant in this concentration range. (27) This allows the calibration of
emission detection instruments with different sensibilities, also being able to calibrate
instruments which measure ultra-low CL emissions like luminometers and similar
apparatus. The hydrogen peroxide concentration must always be in a 100 fold excess
relative to the luminol concentration, in order to guarantee complete consumption of
luminol, the limiting reagent. All solutions, except the luminol stock solution, have to
be prepared immediately before the experiment. Furthermore, data acquisition should be
initiated before the addition of the hydrogen peroxide solution, to ensure the
measurement of the initial emission intensity of the fast reaction, which lasts typically
for a reaction time of about 200 s. Additionally, in the calibration of highly sensitive
detection instruments the light emission observed upon mixing hydrogen peroxide and
hemin (in the absence of luminol) has to be measured in control experiments and this
integrated light emission be discounted from the emission of the complete system. For
very low luminol concentrations (highly sensible detection devices) the hydrogen
peroxide as well as the hemin concentrations should be diminished proportionally to the
luminol concentration.
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(iii)
Calibration
relative
to
3-adamantylidene-4-(3-tert-butyl-dimethylsi-
loxyphenyl)-4-methoxy-1,2-dioxetane (13,28)
The group of Matsumoto uses the chemiluminescence quantum yield for 3adamantylidene-4-(3-tert-butyl-dimethylsiloxyphenyl)-4-methoxy-1,2-dioxetane as a
standard for the determination of the chemiluminescence quantum yields of other 1,2dioxetane derivatives. The chemiluminescence quantum yields obtained in the induced
decomposition of 3-adamantylidene-4-(3-tert-butyl-dimethylsiloxyphenyl)-4-methoxy1,2-dioxetane in dimethylsulfoxide (CL = 0.290 E mol-1) and acetonitrile (CL = 0.116
E mol-1) have been determined by Trofimov et al., utilizing the Hastings-Weber
scintillation cocktail as standard. (13)
The calibration procedure consists in the addition of 1.0 mL of the 1,2-dioxetane
stock solution (1.0 10-5 mol L-1) to a quartz cell containing 2.0 mL of a
tetrabutylammonium fluoride (1.0 10-2 mol L-1) solution in dimethylsulfoxide, at 25oC
in a spectrometer, to initiate the reaction and start the emission intensity measurement.
The total light emission obtained during the induced decomposition of the sample 1,2dioxetane is then compared to that obtained for the standard 1,2-dioxetane derivative in
the same conditions, allowing the calculation of the chemiluminescence quantum yields
for new 1,2-dioxetanes.(28)
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