Singlet-oxygen reactions sensitized on solid surfaces of lignin or titanium dioxide: Product studies from hindered secondary amines and from lipid peroxidation

Article abstract of DOI:10.1139/v02-199

Product analyses and kinetic methods were used to determine the role of singlet oxygen in lignin-catalyzed oxidations of organic substrates. Method A used the ESR analysis of nitroxide radicals formed by singlet oxygen (Type II) on 2,2,6,6-tetramethylpiperidine, 1, or tetramethylpiperidone, 2. Method B used HPLC analysis of the 9- and 13-linoleate chain hydroperoxides formed on oxidation of methyl linoleate to distinguish free-radical peroxidation (Type I) from singlet-oxygen oxidation (Type II) on the basis of different cis,trans (kinetic) to trans,trans (thermodynamic) product ratios. Applications of method A to solid dispersions of lignin or titanium dioxide (TiO₂, a known singlet-oxygen sensitizer) indicated singlet-oxygen reactions. In addition to the nitroxide triplet, irradiation of lignin produces a persistent broad signal in the solid attributed to phenoxyl radicals. Benzophenone and 3,5-di-tert-butyl-ortho-benzoquinone, 5, coated on silica gel were used as models to compare the effects of irradiating such compounds on the products and kinetics of methyl linoleate oxidation. Benzophenone acted as an initiator, giving free-radical peroxidation, whereas 5 or lignin coated with methyl linoleate acted as singlet-oxygen sensitizers, according to both product studies (method B) and the kinetic order in oxygen consumption during UV photolysis. Photolysis of phase-separated sensitizer (TiO₂ or lignin) and substrate (methyl linoleate) resulted in typical singlet-oxygen products. These results indicate that singlet oxygen plays a significant role in the photo-yellowing of high-lignin-content wood pulps.

Full text of DOI:10.1139/v02-199

457
Singlet-oxygen reactions sensitized on solid
surfaces of lignin or titanium dioxide: Product
studies from hindered secondary amines and from
lipid peroxidation
L.R.C. Barclay, M.-C. Basque, and M.R. Vinqvist
Abstract: Product analyses and kinetic methods were used to determine the role of singlet oxygen in lignin-catalyzed
oxidations of organic substrates. Method A used the ESR analysis of nitroxide radicals formed by singlet oxygen
(Type II) on 2,2,6,6-tetramethylpiperidine, 1, or tetramethylpiperidone, 2. Method B used HPLC analysis of the 9- and
13-linoleate chain hydroperoxides formed on oxidation of methyl linoleate to distinguish free-radical peroxidation
(Type I) from singlet-oxygen oxidation (Type II) on the basis of different cis,trans (kinetic) to trans,trans (thermody-
namic) product ratios. Applications of method A to solid dispersions of lignin or titanium dioxide (TiO2, a known sin-
glet-oxygen sensitizer) indicated singlet-oxygen reactions. In addition to the nitroxide triplet, irradiation of lignin
produces a persistent broad signal in the solid attributed to phenoxyl radicals. Benzophenone and 3,5-di-tert-butyl-
ortho-benzoquinone, 5, coated on silica gel were used as models to compare the effects of irradiating such compounds
on the products and kinetics of methyl linoleate oxidation. Benzophenone acted as an initiator, giving free-radical per-
oxidation, whereas 5 or lignin coated with methyl linoleate acted as singlet-oxygen sensitizers, according to both prod-
uct studies (method B) and the kinetic order in oxygen consumption during UV photolysis. Photolysis of phase-
separated sensitizer (TiO2 or lignin) and substrate (methyl linoleate) resulted in typical singlet-oxygen products. These
results indicate that singlet oxygen plays a significant role in the photo-yellowing of high-lignin-content wood pulps.
Key words: lignin, singlet oxygen, mechanism, peroxidation, products.
Résumé : Faisant appel à des analyses de produit et à des méthodes cinétiques, on a déterminer le rôle de l’oxygène
singulet dans les réactions d’oxydation catalysées par la lignine de substrats organiques. La méthode d’analyse A uti-
lise la RPE des radicaux nitroxydes formés par l’action de l’oxygène singulet (Type II) sur la 2,2,6,6-tétraméthyl-
pipéridine (1) ou la tétraméthylpipéridone (2). La méthode B est basée sur l’analyse par CLHP des hydroperoxydes des
chaînes 9- et 13-linoléates qui se forment par oxydation du linoléate de méthyle; sur la base des divers rapports de
produits (cis, trans (cinétique)) par rapport à (trans, trans (thermodynamique)), elle permet de distinguer entre la per-
oxydation radicalaire (Type I) et l’oxydation à l’aide d’oxygène singulet (Type II). Les applications de la méthode A à
des dispersions solides de lignine ou de dioxyde de titane, TiO2 bien connu comme sensibilisateur d’oxygène singulet,
indiquent qu’elles se produisent par des réactions d’oxygène singulet. En plus du triplet nitroxyde, l’irradiation de la li-
gnine produit dans le solide un large signal persistant qui est attribué aux radicaux phénoxyles. Des études modèles ont
permis de comparer les effets sur la nature des produits et sur la cinétique de l’oxydation du linoléate de méthyle de
l’irradiation de couches sur du gel de silice avec de benzophénone ou avec de la 3,5-di-tert-butyl-ortho-benzoquinone
(5). La benzophénone agit comme initiateur d’une peroxydation radicalaire alors que, sur la base des études de produits
(méthode B) et de l’ordre cinétique de la consommation d’oxygène au cours de la photolyse UV, le composé 5 et la li-
gnine recouverte d’une couche de linoléate de méthyle agissent comme sensibilisateurs d’oxygène. La photolyse de
photosensibilisateurs, TiO2 ou lignine, en phases séparées du substrat, le linoléate de méthyle, conduisent à la forma-
tion des produits typiques de l’action d’oxygène singulet. Ces résultats indiquent que l’oxygène singulet joue un rôle
significatif dans le photojaunissement des pulpes de bois contenant de fortes proportions de lignine.
Mots clés : lignine, oxygène singulet, mécanisme, peroxydation, produits.
[Traduit par la Rédaction] Barclay et al. 467
Received 3 July 2002. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 16 April 2003.
Dedicated to Professor Don Arnold for his contributions to chemistry.
L.R.C. Barclay1, M.-C. Basque, and M.R. Vinqvist. Department of Chemistry, Mount Allison University, Sackville,
NB E4L 1G8, Canada.
1Corresponding author (e-mail: rbarclay@mta.ca)
Can. J. Chem. 81: 457–467 (2003)
doi: 10.1139/V02-199
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provided a means of establishing the pathways; namely,
free-radical oxidation (Type I) or singlet-oxygen reaction
(Type II). (d) Phase-separated oxidation: A phase-separated
system was used to mimic the natural lignin–air situation. In
this technique, the substrate, methyl linoleate, is separated
from the sensitizer by a narrow air gap. Product profiles
(HPLC) of linoleate hydroperoxides are compared from irra-
diation experiments using, as sensitizers, titanium dioxide,
benzophenone, and lignin. Structures for the main organic
compounds employed are shown in Fig. 2.
Introduction
Products from Canada’s wood-pulping industry are the
country’s major export. The use of thermal mechanical pulps
has provided more than 50% of the fibre for Canadian paper
products (1). The high lignin content of these products has
resulted in undesirable oxidative photo-yellowing, which can
reduce the value of these products. Consequently, the inhibi-
tion of such yellowing is of major interest. The excited state
of oxygen, singlet oxygen 1∆g, is known to rapidly oxidize a
wide variety of organic substrates (2 and refs. therein) and
may be involved in photo-yellowing of thermal mechanical
pulps. Singlet oxygen produced by dye sensitization sepa-
rately from the substrates is known to react readily with
lignin model compounds (3–8), lignin (9), high-yield pulps
(10–12), and cellulose (13, 14). While these studies seem to
implicate singlet oxygen as a possible mechanism for oxida-
tive yellowing of lignin, controversy has continued about its
significance in this process (5, 10, 11).
Lignin is a complex polymer of phenylpropyl units cross-
linked and highly substituted by ether, carbonyl, hydroxyl,
and methoxyl groups, as illustrated schematically in Fig. 1.
Consequently, lignin is a paradox, in that the hydroxyl
groups provide antioxidant activity against Type I free-
radical oxidation, as observed for lignin-type monomers (15)
and even in lignin itself (16); however, at the same time, the
aromatic carbonyl chromophores could act as singlet-oxygen
sensitizers and initiate singlet-oxygen (Type II) oxidations.
Earlier studies were limited by the lack of evidence for
singlet-oxygen formation directly on lignin itself as its own
sensitizer. Some evidence for the direct formation of singlet
oxygen on lignin was given in our report, which showed that
the light-induced, self-initiated oxidation of lignin was
quenched by sodium azide, a known singlet-oxygen quencher
(2). We now report in more detail on the issue of the role of
singlet oxygen in lignin photo-oxidation. The structural
complexity of lignin and the fact that lignin chromophores
react readily with singlet oxygen (3–9) requires a multifac-
eted approach to the possible formation of singlet oxygen on
lignin. Accordingly, we approached this problem in several
ways, as follows. (a) Product studies: Two known methods
of product analysis were used for gathering evidence of a
singlet-oxygen reaction: (i) electron spin resonance (ESR)
detection of nitroxide radicals formed by singlet-oxygen oxi-
dation of hindered secondary amines and (ii) product pro-
files by high performance liquid chromatography (HPLC) of
peroxidation of the lipid (methyl linoleate) by singlet oxy-
gen compared with free-radical peroxidation. (b) Solid dis-
persions: A comparison was made of the products formed
using a dispersion of titanium dioxide, a known singlet-
oxygen sensitizer, with products formed by photolysis of
lignin dispersions. (c) Kinetic studies: The kinetic orders in
light intensity were determined for oxidation of methyl lino-
leate on silica gel in water catalyzed by the ketones, benzo-
phenone (BP), and 3,5-di-tert-butyl-ortho-benzoquinone
(DTBQ). The latter was selected as a simple lignin model
compound because ortho-quinones are formed during the
early photochemistry of mechanical pulps (17). These results
were compared with the kinetics of oxidation of methyl lino-
leate “sensitized” by aqueous dispersions of lignin. The ki-
netic data and product analyses of linoleate peroxidation
Results
Product studies from singlet-oxygen oxidation of
hindered secondary amines or methyl linoleate
Product studies of (i) oxidation of hindered secondary
(sec.) amines and (ii) peroxidation of methyl linoleate were
carried out initially in solution to evaluate the employed
methods and for comparison with the more complex hetero-
genous systems containing lignin. The conversion by singlet
oxygen of the hindered sec. amines, 2,2,6,6-tetramethyl-
piperidine (TMP, 1) and its 4-oxo derivative (TMPO, 2), to
the corresponding nitroxide radicals, 3, detected by ESR,
have been used for many years and under various conditions
as evidence of a singlet-oxygen reaction (18–30). The method
is simple and very sensitive to ESR detection of the stable
nitroxide radical. However, there are conflicting literature re-
ports on its use. For example, there is at least one report that
states that the TMP reaction with singlet oxygen “is highly
specific” since it does not react with the superoxide anion
nor with the hydroxyl radical (29), while others reported that
these amines, 1 and 2, are useful tools “for trapping hydroxyl
radicals” (30).
We found that great care must be exercised when using
the oxidation of these amines as evidence of singlet oxygen
because nitroxide radicals are often present in commercial 1
and 2 (19, 25). For such work, the following precautions
should be observed: (a) the traces of nitroxide radicals
should be removed before commencing the singlet experi-
ment to avoid ambiguous results. We found a convenient
method to do this is to vortex stir the amine dissolved in
hexane with aqueous hydrazine; (b) parallel control experi-
ments should be done with a singlet-oxygen quencher;
(c) the ESR signals observed should be intense, and the
amount of conversion from the amine to nitroxide radical
measured by integration of the ESR spectra. A typical exam-
ple of the oxidation of TMP using a known singlet-oxygen
sensitizer, methylene blue, and product studies by ESR is il-
lustrated in Fig. 3b. This sample was not pretreated with
hydrazine, and a trace of nitroxide radical is observed at
higher receiver gain (Fig. 3a). However, the conversion on
sensitization (approximately 2%) to the typical nitroxide
triplet (hyperfine nitrogen coupling aN = 16.0 gauss) in
agreement with literature values (23, 29), indicates reaction
by singlet oxygen.
Product distribution of the hydroperoxides from peroxidation
of the lipid, methyl linoleate (Fig. 2, structure 10), is a use-
ful method to distinguish between free-radical (Type I) and
singlet-oxygen (Type II) reactions. Earlier reports described
the separation and identification of the various isomers and
related these to the two mechanisms involved (31–34). Type
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459
Fig. 1. Schematic illustration of the lignin polymer.
I reactions proceed by initial H-atom abstraction from posi-
tion 11 on the linoleate chain, rapid reaction of the derived
carbon-centered radical with oxygen, and rearrangement of
the peroxyl radicals, giving a distribution between the conju-
gated 9- and 13-substituted oxidation products, two with the
cis,trans configuration (kinetic products 6 and 8) and two
with the trans,trans configuration (thermodynamic products
7 and 9). A recent discovery (35) found that the bis-allylic
11-hydroperoxide is the kinetic product initially formed in
the presence of α-tocopherol, a powerful H-atom donor;
however, this product was not detected in our experiments.
These products are usually analyzed as their corresponding
alcohols (Fig. 2) that are formed on reduction of the hydro-
peroxides. On the other hand, the singlet-oxygen reaction
does not involve free radicals. Whether singlet oxygen reacts
with linoleate by a concerted ′ene reaction or its reaction
possibly involves other intermediates (36), the reaction is
kinetically controlled, producing an excess of the cis,trans
over the trans,trans isomers (e.g., a high cis,trans to
trans,trans ratio). So, a significantly higher cis,trans to
trans,trans ratio is expected from a Type II reaction on
linoleate compared with a Type I for the same linoleate con-
centration, bearing in mind that this ratio depends on the H-
atom donating ability of the medium for Type I reactions
(37, 38). In addition, the less selective singlet-oxygen reac-
tion gives products of reaction at the 10 and 12 positions of
linoleate, leading to two nonconjugated isomers, a total of 6
hydroperoxides (2, 31–34). These two pathways, Type I and
Type II, are outlined in Scheme 1. An example singlet-
oxygen product formation from the photolysis of methyl
linoleate sensitized by methylene blue is shown in Fig. 4. In
this case the cis,trans ratio was found to be 14.2 at 2.8% ox-
idation of linoleate. The HPLC method of separation and de-
tection of the isomers at 234 nm shows the relative
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Can. J. Chem. Vol. 81, 2003
Fig. 2. Structural formulas of the main compounds used.
composition of the conjugated isomers, but the non-
conjugated ones will actually be more concentrated than
they appear in the chromatogram, since they do not absorb
as strongly as the conjugated isomers at 234 nm. Our results
focus mainly on the conjugated products and their relative
yields (e.g., the cis,trans to trans,trans ratios).
used, contrasting these with the products formed from TMP
(if any) when solid lignin was used.
A typical experiment using TiO2 dispersed in methylene
chloride for photo-oxidation of TMP is shown in Fig. 5. The
triplet, which formed only in the presence of TiO2, is attrib-
uted to oxidation via singlet oxygen (Fig. 5b). This proce-
dure was then applied to dispersions of solid lignin in solvents.
Figs. 5c and 5d show the results of irradiation of lignin in
methylene chloride containing TMP and a parallel experi-
ment in the presence of the singlet-oxygen quencher 1,4-
diazabicyclo[2.2.2]octane (Dabco). There was no nitroxide
signal in the presence of the quencher (Fig. 5c), whereas the
strong triplet appeared in its absence (Fig. 5d). In addition, a
quantitative study of the radical concentration showed that it
increased with time, as illustrated in Fig. 6. In a similar ex-
periment, we found that extended irradiation of TMP in
lignin–hexane dispersions (not shown) gave a 10% conver-
sion to the nitroxide radical.
Sensitizations on solid dispersions: Titanium dioxide
and lignin
Photolyses involving excited states and intermediates on
solid surfaces are of continuing interest and the subject of
several reviews (39–42). Solid titanium dioxide, like lignin
(presumably), is classified as a reactive surface (40), since it
is a known singlet-oxygen sensitizer (although despite that,
it is also commonly used in cosmetic sunscreens). Irradiation
of TiO2 with 320–400 nm light causes electronic excitation
from a valence band to a conduction band, which allows en-
ergy transfer to oxygen (27). Because of the expected com-
plexity of the excited state chemistry on the solid lignin
polymer, we employed a comparative study of products
formed from TMP when titanium dioxide sensitization was
The ESR spectrum resulting from relatively short irradia-
tion of lignin dispersed in hexane in the presence of 4-
OXOTMP (2) was more complex, as shown in Fig. 7a, and
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461
Fig. 3. ESR spectra from irradiation in PR–350 of tetramethylpi-
peridine (TMP), 1, 7.7 mM, in methylene chloride for 15 min.
(a) Without sensitizer, receiver gain (R.G.) of 5.0 × 105; (b) with
9.2 × 10–8 mol methylene blue, R.G. = 1.25 × 105, [R·] =
0.14 mM. The peak on the high field part of the triplet, which
appears here and in other spectra, is due to a permanent cavity
signal.
where kp and 2kt are the rate constants for chain propagation
and termination, respectively, and Ri is the rate of free-radical
initiation. For photo-initiated H-atom abstraction by the n,π*
triplet state of ketones (Type I mechanism), the kinetic order
in oxygen consumption is expected to be half-order in light
intensity, since this controls the Ri, and this was observed
earlier for benzophenone photo-initiated peroxidation of
linoleate in heterogeneous aqueous micelles (44). On the
other hand, the kinetic order for the peroxidation of linoleate
photo-initiated by the coloured lignin model quinone (5) was
approximately unity, indicative of a change in mechanism to
Type II with this quinone as sensitizer (2). With these earlier
results in mind, we determined the kinetic order in light
intensity — together with the trends in product profiles —
for oxidation of methyl linoleate initiated by benzophenone
and by 5 in aqueous dispersions, for comparison with similar
data using lignin in water. The ketones and methyl linoleate
were used as films on silica gel since this is classed as a
non-reactive surface (40); moreover, benzophenone is an ef-
fective H-atom abstractor when adsorbed on silica gel (45).
Therefore, these two compounds, benzophenone and 5, were
expected to provide different kinetic orders of oxygen uptake
in the oxidation of methyl linoleate. In an experiment using
lignin, the lipid was evaporated directly onto the lignin. The
results of typical experiments are shown in Fig. 8. The ex-
periment using benzophenone (Fig. 8a) gave a kinetic order
of 0.55, indicative of a Type I pathway, while with the
quinone (5) the kinetic order (Fig. 8b) was significantly
higher at 0.90. The oxygen uptake in the benzophenone –
methyl linoleate experiment was inhibited by the active wa-
ter-soluble antioxidant Trolox, 11, which gave a distinct in-
duction period (not shown) similar to that observed earlier
for photolysis with this combination in micelles (44). It ap-
pears that Trolox can trap chain-propagating peroxyl radicals
generated on the methyl linoleate – silica gel surface, much
like it does in other media. In contrast, Trolox gave no in-
duction period during the photo-reaction combination of 5 –
methyl linoleate. Lignin was also an effective photocatalyst
for lipid oxidation and gave a kinetic order of 0.84 (Fig. 8c),
similar to that observed with the quinone.
Product studies were carried out on oxidations of methyl
linoleate under similar conditions to those in the kinetic
runs. Typical product profiles from reactions using benzo-
phenone and quinone, 5, are shown in Figs. 9a and 9b.
These product profiles are clearly different: the reaction us-
ing benzophenone gave the four conjugated isomers with a
cis,trans to trans,trans isomer ratio of ca. unity (Fig. 9a),
while six isomers were formed with a cis,trans to trans,trans
ratio of ca. 32 when using quinone in the photolysis
(Fig. 9b). The different kinetic orders together with different
product profiles allow one to distinguish between the two
possible mechanisms in these heterogenous systems: a
Type I free-radical oxidation initiated by H-atom abstrac-
tion by excited benzophenone vs. a Type II singlet-oxygen
reaction sensitized by the quinone. Product analysis from
oxidation of methyl linoleate on lignin (not shown) also
showed an excess of the cis,trans isomers, but the
chromatograms were very complex in the region where the
trans isomers appear, preventing reliable calculations of the
isomer ratios. However, these qualitative product studies,
together with the kinetic order experiment, indicate the par-
consisted of a very broad signal superimposed on the ni-
troxide triplet. The Lande g-factor for the broad signal was
estimated to be g = 2.004–2.005 with reference to that of the
nitroxide radical, g = 2.006 (29). This g-factor for the broad
signal is in the range of a wide variety of phenoxyl radicals
substituted by alkyl, hydroxyl, methoxy, and keto groups,
where g = 2.003–2.005 (43). The separated solution phase
shows the spectrum of the nitroxide radical more clearly
(Fig. 7b). The broad signal was also very persistent, and it
could be observed in the lignin that was separated from
TMP by filtration. Separate irradiation of lignin alone in
hexane also produced this lignin signal (Fig. 7c), which on
dissolution of the lignin in dioxane disappeared and did not
reappear on further irradiation of the lignin–dioxane solu-
tion. It is also interesting to note that with irradiation of
lignin, under these conditions but in the presence of Dabco,
the broad signal appeared but not the nitroxide triplet (not
shown).
Kinetic and product studies of methyl linoleate
peroxidation sensitized by lignin in water: Comparisons
with sensitizations by benzophenone and by 3,5-di-tert-
butylbenzoquinone, 5
The kinetics of free-radical lipid peroxidation is known to
follow the classical rate law for autoxidation (44) repre-
sented by the following equation (eq. [1]):
[1]
–d[O2]/dt = kp/(2kt)1/2 × [lipid] × Ri1/2
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Can. J. Chem. Vol. 81, 2003
Scheme 1. Free radical (Type I) and singlet-oxygen (Type II) pathways for lipid peroxidation.
they are together in solution during singlet-oxygen oxida-
tions, such as competing H-atom abstraction by the sensitizer
or electron transfer reactions. In addition, the lifetime of sin-
glet oxygen is much longer in the gas phase than in solution
(46), which should make the reaction more efficient.
Fig. 4. HPLC trace of photo-initiated oxidation products in air
from methyl linoleate, 0.50 M in chloroform, with methylene
blue, 1.11 × 10–2 M, using a 1000 W visible lamp for 10 min.
Peaks labeled 6, 7, 8, and 9 are the 13 cis,trans, 13 trans,trans;
9 cis,trans, and 9 trans,trans isomers, respectively (see Fig. 2).
The sample was 2.8% oxidized, giving a cis,trans to trans,trans
product ratio = 14.2. Peaks marked by vertical arrows are non-
conjugated isomers (see text). In this and other HPLC separa-
tions, PMA = 4-methoxyacetophenone, used as an internal
standard for retention times and total hydroperoxides formed.
A simple apparatus was constructed for some experiments
with the sensitizer and substrate separated 1 to 2 mm by an
air gap (Fig. 10), a space that is expected to allow for diffu-
sion of singlet oxygen across the gap (46, 47). The sensitizer
was coated on the bottom of the top filter-petri dish either as a
film (e.g., benzophenone) or as a powder on double-sided
tape. The sensitizer and substrate were then separated by two
microscope slides, and the assembly was mounted on a cold
plate and moved into the photoreactor. In practice, our proce-
dure had limitations because the coating of sensitizers often
“screened” much of the light, making the process less effi-
cient so that long irradiation times were needed, and this can
result in thermal or photo-rearrangements of initially formed
hydroperoxides in the products (see Discussion section).
We used benzophenone as a typical photo-initiator to test
the effect of the PS procedure as compared with results ob-
tained using a homogeneous solution. In the latter case, the
classical free-radical reaction (Type I) is known to predomi-
nate (44), and this was confirmed in the present case, since
the isomer distribution showed a predominance to trans,trans
isomers (cis,trans to trans,trans ratio = 0.44, Fig. 11a). The
PS experiment required longer irradiation times to obtain
comparable product buildup for HPLC analysis, and typi-
cally the product profiles (Fig. 11b) from these experiments
were quite different from reactions in solution, showing all
six isomers and an excess of the cis,trans isomers (cis,trans
to trans,trans ratio = 9.1).
ticipation of singlet oxygen in the lignin – methyl linoleate
photolysis.
Oxidations by separation of the sensitizer and substrate
(phase separation, PS)
Physical separation of the sensitizer from the substrate
should eliminate complications that are always present when
To evaluate the effect that lignin might have in the PS pro-
cedure, experiments were carried out with titanium dioxide
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463
Fig. 6. Plot of nitroxide concentration vs. time for irradiation of
TMP, 1, 7.7 mM, with a dispersion of lignin, 15 mg, in methy-
lene chloride in PR–350.
Fig. 5. ESR spectra from irradiation in air by PR–350 of methy-
lene chloride solutions of TMP treated with hydrazine before ir-
radiation. (a) With TMP, 0.40 M, and irradiated for 3 h, R.G. =
2.0 × 105; (b) TMP, 0.40 M, in the presence of titanium dioxide
dispersion, irradiated for 45 min., R.G. = 4.0 × 104; (c) with
TMP, 7.7 mM, Dabco quencher, 0.34 M, and 20 mg of lignin
dispersion, irradiated for 40 min., R.G. = 5.0 × 105; (d) TMP
and lignin, as in (c) but without Dabco, irradiated for 40 min.,
R.G. = 1.5 × 105. The additional peak on the high field side of
the triplet, which appears here and in other spectra, is due to a
permanent cavity signal.
solution = 0.44), reflecting the effect of a higher substrate
concentration for essentially neat substrate on silica gel.
Experimental
Materials and preparations
The following chemicals used were the purest grade from
Aldrich: 2,2,6,6-tetramethylpiperidine (TMP), 2,2,6,6-tetra-
methyl-4-piperidone (TMPO), 1,4-diazabicyclo[2.2.2]octane
(Dabco), 3,5-di-tert-butyl-ortho-benzoquinone, 4-methoxy-
acetophenone, benzophenone, titanium dioxide (Anatase- pow-
der), N,N-dimethyl-p-phenylene–2HCl (DMP–HCl), triphenyl-
phosphine, and Trolox. Methyl linoleate (>99%) was obtained
from Nu-Chek-Prep and silica gel (100–200 mesh) from
Mallinckrodt. The lignin used was milled from unbleached
softwood at Paprican, Pointe Claire, Quebec, Canada.
The TMP and TEMPO (sublimed in vacuo) were normally
purified to remove nitroxide signals just before use in each
experiment (see text). In a typical purification, an equal vol-
ume of TMP or TMPO and aqueous hydrazine (35%, Aldrich)
in the solvent used (methylene chloride or hexane) were vor-
tex stirred for 2–5 min. The mixture was centrifuged to sepa-
rate the organic layer, which was dried over sodium sulfate
and purged with argon, and the sample was checked for ESR
signals before use. Commercial silica gels were washed free
of traces of free metal ions before use. For this purpose the
material was washed at least three times with distilled water
then twice with methanol, and the samples were oven-dried
overnight. Traces of hydroperoxides were removed from
commercial methyl linoleate by column chromatography on
silica gel under argon, and fractions free of hydroperoxides
(by TLC analysis, as detected by a DMP–HCl spray) were
used in experiments. Alternately, it was convenient to pre-
pare hydroperoxide-free solutions of methyl linoleate by vor-
tex stirring a solution of the ester in distilled hexane with
silica gel (nominally: 200 mg of ester in 50 mL hexane and
70 mg of silica gel). The solutions were checked for hydro-
peroxides by UV absorption at 234 nm (conjugation) and by
TLC before use.
powder and with lignin powder for a comparison of the
product profiles in the oxidation of methyl linoleate. These
results are shown in Fig. 12. For similar relative conversions
of the methyl linoleate (ca. 6%), the product profiles of the
isomers are quite similar, with cis,trans to trans,trans ratios
of 10.4 and 12.7 for the titanium dioxide sensitizer
(Fig. 12a) and lignin as sensitizer (Fig. 12b), respectively.
Both reactions appear to involve significant oxidation by
singlet oxygen. This result indicates the specific advantage
of phase separation to study a singlet-oxygen reaction. When
the benzophenone and methyl linoleate were on separate sil-
ica gel surfaces but irradiated in the same glass vessel the
product distribution (Fig. 9a) was similar to that found for a
homogeneous solution (Fig. 11a). Presumably, this is be-
cause benzophenone and methyl linoleate were in close con-
tact on the two silica gel surfaces, resulting in a process
similar to a homogeneous system but with formation of a
higher cis,trans to trans,trans ratio (on silica = 0.91 cf. in
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Can. J. Chem. Vol. 81, 2003
Fig. 7. ESR spectra from irradiation with a 200 W Hg arc lamp
in air of hexane dispersions of lignin, 10 mg, in the probe of the
spectrometer. Samples were purged with argon before recording
spectra. (a) With TMPO, 6.7 mM, irradiated for 5 min., R.G. =
5.0 × 105; (b) the spectrum of the solution after separation of the
lignin, R.G. = 2.0 × 105; (c) Irradiation of lignin dispersion
alone in hexane for 12 min., R.G. = 2.0 × 105.
Fig. 8. Plots of kinetic orders of oxygen uptake vs. light inten-
sity for photo-initiated oxidation of methyl linoleate on silica gel
in water at 30°C; comparing the effects of the ketones, benzo-
phenone, DTBQ, 5, and lignin. (a) Methyl linoleate, 54 µm, on
125 mg silica gel with benzophenone, 5.6 µm, on 100 mg silica
gel, irradiated with 200 W Hg arc lamp; (b) methyl linoleate,
56 µm, on 112 mg silica gel with DTBQ, 5, 8.7 µm, on 185 mg
silica gel irradiated with a 1000 W visible lamp; (c) methyl lino-
leate, 54 µm, evaporated from methylene chloride on lignin,
92 mg, and irradiated with a 200 W Hg arc lamp.
aration (PS) method; it was mixed with silica gel (3 g per
500 mL) to hold the sample in the sample well close to the
sensitizer (see Fig. 10 and text). In these cases, the silica gel
was washed with methanol to remove the adsorbed oxidation
products for reduction and HPLC analyses.
Photolysis methods
Photolyses were carried out using pyrex filters. Irradia-
tions at 350 nm were done in a photoreactor constructed in
the laboratory of J.C. Scaiano, University of Ottawa, Can-
ada, consisting of an air-cooled metal box (30 cm high ×
35 cm × 35 cm) fitted with eight 25 W, 350 nm lamps, a
prototype of a Luzchem Photoreactor, Inc. A cold plate
(Stirr Kool Inc.), fitted into the reactor, permitted samples to
be cooled to between 0 and –10°C. This method, used for
350 nm photolysis, is referred to as PR–350 in the figure
legends. A 200 W Oriel Hg–Xe lamp was used for
photolyses undertaken in the probe of an electron spin reso-
nance spectrometer (ESR); an Oriel 1000 W quartz–tungsten
lamp was used for those experiments employing coloured
dyes as sensitizers, such as methylene blue or 3,5-di-tert-
butyl-ortho-quinone. A solution of the ester in an inert sol-
vent (e.g., hydrocarbons, heptadecane, or dodecane) was used
in the photo-irradiation of methyl linoleate by the phase sep-
Product analyses
Electron spin resonance (ESR)
ESR spectra were obtained on a Varian E-3 EPR Spec-
trometer operated generally at a power level of 3 to 3.2 mW
and a modulation amplitude of 4 G to optimize signals.
Quantitative analysis of nitroxide signals used double inte-
gration of the absorption curves and diphenylpicrylhydrazyl
as a concentration standard (48). The integrations were cor-
rected for ESR cavity response by the signal of a ruby in-
stalled in the probe.
HPLC method
The procedures for reduction of methyl linoleate hydro-
peroxides by triphenylphosphine and HPLC analysis of the
resulting isomeric alcohols and their identification was de-
scribed previously (2, 37). 4-Methoxyacetophenone was
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Barclay et al.
465
Fig. 9. HPLC traces of oxidation products of methyl linoleate on
silica gel in water at 30°C; comparing the effects of benzophe-
none and DTBQ, 5. The quantities of methyl linoleate, benzo-
phenone, and DTBQ were the same as in Fig. 8. (a) Benzophe-
none and irradiation with a 200 W Hg arc lamp. The cis,trans to
trans,trans product ratio was 0.91 at 6.6% oxidation; (b) DTBQ
and irradiation with a 1000 W visible lamp. The cis,trans to
trans,trans product ratio was 32 at 9.4% oxidation. See Fig. 4
for identification of the HPLC peaks.
Fig. 11. HPLC traces of methyl linoleate oxidation products
showing the result using “initiation” by benzophenone in solution
compared with phase separation of benzophenone as “sensitizer”.
(a) Methyl linoleate, 0.43 M, with benzophenone, 5.5 mM, in
dodecane, irradiated in PR–350 for 1 h, giving 1.6% oxidation of
methyl linoleate at a cis,trans to trans,trans product ratio = 0.44;
(b) methyl linoleate, 0.69 M, in heptadecane and benzophenone
powder, 29 mg, in the PS apparatus (Fig. 10) irradiated for 9 h,
giving 2.2% oxidation at a cis,trans to trans,trans product ra-
tio = 9.1. The identification of the peaks is given in Fig. 4.
Fig. 10. Diagram of apparatus used for the phase separation (PS)
method.
quite conclusive results on the role of lignin in the genera-
tion of singlet oxygen. ESR studies of nitroxides from the
oxidation of secondary amines 1 and 2 indicate singlet-
oxygen generation by the photolysis of dispersions of solid
lignin. A singlet-oxygen quencher inhibits the reaction, and
in the absence of quencher the reaction products, nitroxides,
increase in quantity with time. This ESR method has the ad-
vantage of high sensitivity, but the appearance of nitroxide
radicals alone does not rule out the possible involvement of
Type I reactions occurring simultaneously with singlet-oxygen
oxidation. In this regard, the broad ESR signal produced
during shorter irradiation times on lignin dispersion together
with nitroxide formation is of special interest. Wan and co-
workers observed similar broad, persistent signals (g =
2.0039) on papers from mechanical pulps, attributed them to
phenoxyl radicals formed by H-atom abstraction by excited
chromophores (49), and showed their importance in the
photo-yellowing of high lignin content pulps and paper (50,
51). Our results show that these radicals are formed from the
photo-excited lignin component, presumably by H-atom ab-
straction by excited carbonyl groups. More importantly, the
complex ESR spectrum (Fig. 7a) demonstrates that both
Type I and Type II reactions are initiated from photo-excited
lignin: one type by H-atom abstraction forming phenoxyl
radicals, which are persistent in the polymer matrix, and the
other by energy transfer to oxygen, resulting in nitroxide
used as an internal standard to determine product concentra-
tions, which are reported as “relative concentrations” of the
total 6–9 isomeric compounds (Fig. 2).
Kinetic methods of oxygen uptake
Kinetic studies of oxidation were carried out at 30°C un-
der 760 torr (1 torr = 133.322 Pa) of oxygen on a dual chan-
nel apparatus attached to a pressure transducer, as described
previously (2). The kinetic order in oxygen uptake was de-
termined by inserting seven neutral-density filters on a filter
wheel into the light beam.
Discussion
The combinations of product studies, kinetic measurements,
and phase separations of sensitizer and substrate provide
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466
Can. J. Chem. Vol. 81, 2003
Fig. 12. HPLC traces from oxidation of methyl linoleate using
the PS method to compare the effect of lignin with that of tita-
nium dioxide as “sensitizers”. (a) Methyl linoleate, 0.75 M, in
heptadecane and titanium dioxide powder, 7.2 mg, irradiated for
8 h in PR–350, resulting in 6.2% oxidation and a cis,trans to
trans,trans product ratio = 10.4; (b) methyl linoleate, 0.75 M, in
heptadecane and lignin powder, 9.4 mg, irradiated for 1 h in
PR–350, resulting in 6.3% oxidation and a cis,trans to
trans,trans product ratio = 12.7. The identification of the peaks
is given in Fig. 4.
induced homolysis of the hydroperoxides that accumulate
during prolonged irradiation times, resulting in some com-
peting Type I reaction. In any event, lignin is an active “cat-
alyst” for the oxidation of the lipid, methyl linoleate, on its
surface (Fig. 8c). The reason for the difference in behavior
of the two ketones — as shown by product profiles from the
reactions on silica gel (Fig. 9a and 9b), where benzophenone
causes initiation of a Type I reaction on methyl linoleate
while the ortho-quinone acts as a sensitizer — probably lies
in the nature and energy of two different excited states of
3
these compounds. The (n,π*) of benzophenone is a well-
known, very reactive H-atom abstractor. Although the redox
properties of 5 have been studied in detail, including reduc-
tion by ascorbic acid (52), details on its excited states are
not available. However, the triplet states of 2- to 4-ring aro-
matic ortho-quinones exhibit “inversion” between n,π* and
π,π* triplets where these were not active H-atom abstractors
(53). Similarly, it appears that the excited state of quinone,
5, is not an H-atom abstractor under our conditions but rather
gives energy transfer, forming singlet oxygen.
We are well aware of the limitations of using hydro-
peroxide product studies as evidence for a singlet-oxygen re-
action. In general, secondary photo reactions, especially by
UV light, can cause further reactions on sensitive hydro-
peroxides and complicate the results, as pointed out by oth-
ers (54–56). Consequently, in our results using geometric
product ratios of the cis,trans and trans,trans isomers, it is
not surprising that quite different ratios were observed even
in reactions that apparently proceed via singlet oxygen. Most
ratios were in the range of 9 to 14 (Figs. 4, 11b, 12a, and
12b), and the high ratio combined with the appearance of six
isomers is taken as evidence for a pathway controlled by sin-
glet oxygen, especially when compared with these ratios un-
der conditions recognized to be a Type I reaction (Figs. 9a
and 11a), where the highest ratio was ca. 1. In comparison,
the ratio of ca. 33 when the ortho-quinone, 5, was used as
sensitizer in the visible region (Fig. 9a) is very different and
suggests that the other examples may either indicate second-
ary photo rearrangements or a mixed Type I and Type II
pathway. Triphenylphosphine (TPP) is used to rapidly re-
duce hydroperoxides to alcohols. Since it absorbs only weakly
above 300 nm (57), we considered that photolysis experi-
ments in the presence of TPP would reduce hydroperoxides
as they form and simplify the product analysis. Unfortu-
nately, other reactions of triphenyl phosphine — including
those with excited states of ketones (58, 59) and with singlet
oxygen (60) — actually complicate the system, and this pro-
cedure was abandoned.
Our experiments using lignin indicate that both Type I and
Type II reactions can occur on photolysis. For example, the
observation of two radicals following irradiation of 2 in the
ESR cavity after a comparatively short irradiation time indi-
cates that H-atom abstraction from phenolic hydroxyls (pos-
sibly by excited carbonyl groups) and singlet-oxygen
reactions to generate nitroxide radicals occur simultaneously.
The phase separation method does definitely show that sin-
glet oxygen can be generated by the lignin surface
(Fig. 12b). Overall we conclude that singlet oxygen plays an
important role in the photo-yellowing of high-lignin-content
materials, and consequently, this must be taken into consid-
radical formation by a singlet-oxygen reaction. The lignin–
phenoxyl radicals were not observed in dioxane, probably
owing to rapid radical–radical recombinations in solution.
The use of both kinetic experiments of oxygen uptake and
product studies using methyl linoleate shows that the two
mechanisms of peroxidation depend on the initiator or
sensitizer used. Thus benzophenone, a known, excellent H-
atom abstractor, showed a kinetic order of 0.55 when coated
on silica gel, consistent with initiation by H-atom abstrac-
tion. The results with an ortho quinone were quite different:
the higher kinetic order indicates a change in mechanism
and the HPLC product profile indicates a singlet-oxygen re-
action. As observed before in homogeneous solution (2), ki-
netic orders for singlet-oxygen oxidation are close to unity,
which is expected if one assumes that the oxygen uptake is
directly related to the steady-state concentration of singlet
oxygen. The somewhat lower kinetic orders, ca. 0.9 — actually
observed for oxidations sensitized by the ortho-quinone, 5, a
known singlet-oxygen sensitizer, and for dispersions of lignin
when coated on silica gel — could be due to some photo-
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467
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Acknowledgments
This research was supported by grants from the Mechani-
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the Natural Sciences and Engineering Research Council of
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