New Journal of Glass and Ceramics, 2011, 1, 7-12
doi:10.4236/njgc.2011.11002 Published Online April 2011 (http://www.SciRP.org/journal/njgc)
7
Effect of Aging on Chlorophyll Species Embedded
in Silica Xerogels Matrix
José R. Martínez1*, Erika Espericueta1,2, Gerardo Ortega-Zarzosa1
1Universidad Autónoma de San Luis Potosí, San Luis Potosí, México;
2Centro de Investigación en Materiales Avanzados, Chihuahua, México.
Received February 16th, 2011; revised March 30th, 2011; accepted April 6th, 2011.
ABSTRACT
The spectral fluorescence characteristics of extracts leaves embedded in silica xerogel matrix promoted by aged time
was studied. We obta in a higher PSII stability for extra ct of leaves embedded in xerogel matrix, with concen tration of 8
g / 2 0 ml and water to TEOS molar ratio o f 11, which remain bioactive over a very long period of time, whereas for a
water to TEOS molar ratio of 5 a blue-shift fluor escence is observed indicating the PSII d enaturizing and formation of
fluorescing aggre gat es associated with the act i vat i o n of carot e n oi ds rad i cal s.
Keywords: Fluorescence Spectra, Sol-Gel, Chlorophyll, Extract Leaves, Photosystem II
1. Introduction
Sol-gel has even been used to encapsulate and maintain
the activity of biomolecules. However, currently investi-
gation about organically-modified sol-gels in an effort to
control overall dopant mobility, accessibility, and reac-
tivity are made in the present.
The number of potential applications of organic doped
sol-gel glasses is very large; important possibilities in-
clude optics and electro-optics. Development of these
applications requires a good understanding of the struc-
ture of the doped sol-gel matrices, the properties of the
matrices on the molecular level and the conditions that
the oxide network imposes on the optical properties of
the dopant [1].
Sol-gel technique with the ability to trap photoactive
substances in an inorganic gel glass, has led to some new
applications in nonlinear optics, solid-state tunable lasers,
photochemical hole burning, etc [2,3].
Metalloporphyrin complexes have received much at-
tention owing to their important role in biology, solar
energy conversion, photonics, and catalysis [4]. Spectro-
scopic evidence confirmed stability of the dispersion
over a period of three to four months.
As an optical substance adsorbed into an inorganic
matrix, chlorophyll and composites based in chlorophyll
has been poorly studied, in spite of the interesting appli-
cations of chlorophyll’s optical properties [5-7].
The higher plants are an efficient system to absorb and
transfer the energy through photosynthetic apparatus.
This situation can be used to take advantage to prepare
materials based in extract of plants for eventually design
efficient optical- and electrooptical-based sol-gel encap-
sulated biomolecules [8].
The immobilization of organic pigments on inorganic
media may be useful for the design of energy conversion
devices [9].
Thus, it is necessary to understand the structural evo-
lution of extracted components of leaves embedded in
silica xerogel matrix, as well as the interaction between
the pigments embedded into the inorganic matrix.
The typical fluorescence spectrum of green plants,
embedded or not embedded in xerogel matrix, is in the
range from 650 nm to 780 nm, presenting two maximum
of fluorescence located at about 670 nm and 730 nm
corresponding to chlorophyll a and at about 650 nm and
710 nm corresponding to chlorophyll b [8,10].
All green plants contain chlorophyll a and chlorophyll
b in their chloroplast. In higher plants, chlorophyll a is
the major pigment and chlorophyll b is an accessory
pigment, and the a/b ratio is generally around 3 to 1 [11].
Chlorophyll b differs from chlorophyll a by having an
aldehyde (–CHO) group instead of a methyl group
(–CH3).
The differences in these structures cause that the two
maximum of fluorescence of chlorophyll a are located at
670 nm and 730 nm, while the two maximum of fluores-
cence of chlorophyll b are at 650 nm and 710 nm [10,12].

Effect of Aging on Chlorophyll Species Embedded in Silica Xerogels Matrix
8
The green plants contain the photosystem I and II (PSI,
PSII). A photosystem consist of a peripheral antenna or
ligh-harvesting complex, an inner antenna and the reac-
tion center. The Photosystem II is the principal system in
which the electron transfer in the photosynthesis process
occur.
Chlorophyll is present in the two photosystems, PSI
and PSII, but only photosystem II has additional minor
chlorophyll a/b binding antenna protein. Both photosys-
tems differ in their absorption and fluorescence charac-
teristic. The maximum of fluorescence intensity can be
found for PSI at 735 nm and PSII at 683 nm. At room
temperature it can be assumed that all variable fluores-
cence originates from PSII [10,13].
The photosystem PSII can be monitoring by chloro-
phyll a fluorescence. Thus the observed maximum inten-
sity fluorescence, corresponding to excitation energy
transfer to the photosystem II, is at about 683 nm [8].
Metal porphyrins are molecules present in nature, such
leaves, for example the magnesium porphyrin in chloro-
phyll. The components of the complex extracted of
leaves includes the chlorophyll, which is the principal
component in most green plants, but exist in the Photo-
system II at least 25 different types of protein subunits,
PSII is a large supramolecular pigment-protein complex
embedded in the thylakoid membranes of green plants.
In previous works we show that the samples with ex-
tract of leaves embedded in silica xerogel matrix show
that PSII chlorophyll fluorescence transients is non pre-
sent and the samples show a higher PSII thermostability
for leaves embedded in xerogel matrix than in the green
leaves [8,14].
Under heat treatment, the original bands located at
about 670 and 714 nm observed in the fluorescence
spectrum, are substituted by a blue-shifted band at about
580 nm. These changes can be attributed to the tight en-
vironment around the chlorophyll and to the different
polarity of the environment inside the pores of the silica
matrix, among the conversion of chlorophyll to protein
species, as phaeophytene [15,16].
The large shift is typical for weakening of chlorophyll
– protein interactions in pigment – protein complex
(PPCs). One may assume that the observed spectral
changes are associated with changes of spectral distortion
in the pigments embedded in silica xeroge l matrix during
heat treatment, due to the structural evolution of the ma-
trix.
In this work, by measurements of the PSII fluores-
cence comportment, we report biostability of sol-gel
amorphous bulk samples containing extract of spinach
leaves which is sensitive to concentration of the aggre-
gates.
2. Experimental Procedure
The standard method to extract compound of leaves is by
crude extraction from frozen leaves, washed with water,
by simple grinding and mixing them with ethanol solvent.
For the extraction three arbitrary concentrations, 4 g, 8 g
and 50 g of leaves for 20 ml of ethan ol, corresponding to
low, medium and very high concentration, were used, in
order to obtain information abou t the effect of con centra-
tion in the aging process. The solution of ethanol and
extract was centrifuged at 3000 rpm for 5 minutes and
then filtered with a Wattsman filter nr. 1.
In order to obtain the organic composites of extract of
leaves embedded in xerogel matrix, a precursor material
composed of TEOS, water, and ethanol with pigments
extract of leaves, of spinach was prepared. The set of
samples were prepared using a constant ethanol to TEOS
molar ratio of 4:1 and a molar ratio of water to TEOS (R)
of R = 11 and R = 5. These quantities correspond to a
high water/TEOS ratio, needed to enhance the hydrolysis
to assure a close amorphous structure for the as-prepared
SiO2 powder [17]. The TEOS was dissolved in the etha-
nol with pigments using magnetic stirring for 15 min and
then the water was added to the ethanol-TEOS solution
using magnetic stirring for 10 minutes to form the start-
ing material. The pH of the final solution was 5.
Soft pieces of the gel were obtained after 48 hr. Those
pieces were ground to form a fine powder.
The emission spectra measurements were carried out
in a fluorescence spectrometer by Ocean Optics Inc.
model SF2000 using a reflectance diffuse 45˚ configura-
tion using a double fiber optic component, excited with
an Omnicrome argon ion laser of 532 nm and filter
HNF-532-1.0, in order to obtain the fluorescence contri-
bution due to the photosystem II of samples measured
after gelling and for the same samples measured after 36
months of gelling.
In order to get similar grain size the samples were
ground to form fine powder; the experimental set up to
obtain emission spectra measurement was regularly used
for compared spectroscopy measurement, such NIR,
Raman and fluorescence, obtained in the same experi-
mental condition, in this case by relative intensity fluo-
rescence.
3. Results and Discussion
In order to investigate the structural and fluorescence
change, the set of samples with different extract concen-
tration were irradiate with 532 nm laser using the diffuse
reflectance, the fluorescence results obtained are shown
in the next figures.
Figures 1-3, shows the fluorescence spectra for silica
xerogel with 4 g, 8g and 50 g per 20 ml of extract spin-
ach leave with R = 11, respectively. The as-prepared
samples present the typical chlorophyll a fluorescence
(range from 650 nm to 780 nm) in which no change of

Effect of Aging on Chlorophyll Species Embedded in Silica Xerogels Matrix9
fluorescence characteristic occurred with aged time in-
creasing. For Figures 1 and 3, a blue-shift of the spectra
is observed when the aged time is increased at 36 months
and the maximum fluorescence shift from 678 nm to 669
nm, and 686 nm to 675 nm, after 36 month, respectively,
whereas for Figure 2, the maximum fluorescence is un-
changed maintenance at 678 nm. In the latest case we
obtain a great biostability for su ch formation parameters.
When the ratio R is reduced to R = 5, maintaining the
concentration of 8 g / 20 ml the biostability is break ing as
we can observed in Figure 4. For this sample the aged
effect decomposes the chlorophyll contribution and a
denaturalizati o n occu rs a s we di scuss in next paragra ph.
550 600 650700 750
0 month
36 month
Normalized fl uor esc e nce in t ens ity
Emission wavelength (nm)
Figure 1. Fluorescent emission spectra of spinach leaves,
with concentration of 4 g / 20 ml, embedded in silica xerogel
matrix measured after gelling and after 36 months of gel-
ling.
550 600 650700 750
36 month
0 month
Emission wavelength (nm)
Normalized fluorescence intensity
Figure 2. Fluorescent emission spectra of spinach leaves,
with concentration of 8 g / 20 ml, embedded in silica xerogel
matrix measured after gelling and after 36 months of gel-
ling.
550 600 650 700 750
0 month
36 month
Normaliz ed fluo res cenc e intens i ty
Emission wavelength (nm)
Figure 3. Fluorescent emission spectra of spinach leaves,
with concentration of 50 g / 20 ml, embedded in silica xe-
rogel matrix measured after gelling and after 36 months of
gelling.
550 600 650700 750
0 month
36 month
Normalized fluorescence intensity
Emission wavelength (nm)
Figure 4. Fluorescent emission spectra of spinach leaves,
with concentration of 4 g / 20 ml and water to TEOS molar
ratio of 5, embedded in silica xerogel matrix measured after
gelling and after 36 months of gelling.
Figure 2 shows there is not significant change of
fluorescence of chlorophyll entrapped in silica xerogel up
to 36 months, revealing unchanged in chlorophyll struc-
ture, in samples where extracted leaves of spinach were
embedded in silica xerogel, thus the chlorophyll a mo l e -
cules remain structurally same in silica xerogel over a
very long period of time. In particular the maximum po-
sition of fluorescence of the PSII unchanged in terms of
sol-gel aging time. This time period is very long com-
pared with time, about 3 months, reported in work where
chlorophyll a molecules was entrapped in silica gel
nanomatrix [16].
This results contrast with the case of barley leaves
embedded in silica xerogel matrix where a 10 nm blue
shift of the red emission maximum after aging for about
12 months was observed [8].

Effect of Aging on Chlorophyll Species Embedded in Silica Xerogels Matrix
10
In conclusion extract of spinach leaves with concen-
tration of 8 g / 20 ml and R = 11 embedded in silica xe-
rogel matrix present a higher PSII biostability, remaining
bioactive over very l ong periods of time.
Thus the formation of biostable compound, for very
long aged times, of chlorophyll species embedded in sil-
ica xerogel can be obtained by controlling concentration
of extract leave and ethanol to TEOS and water to TEOS
molar ratios.
In Figure 1 where a 9 nm shift of maximum fluores-
cence is observed, which can be associated with changes
of spectral distortion in the pigments embedded in silica
xerogel matrix during the aged, due to the structural
evolution of the matrix. The shift is attributed also to
changes of the tight environment around the chlorophyll
and to the different polarity of the environment insid e the
pores of the silica matrix, among the conversion of chlo-
rophyll to protein species, as phaeophytene [15,16]. In-
dicate us that, among the previous assignment for the
fluorescence blue shift, denaturations of chlorophyll spe-
cies occur with a increasing of chlorophyll b respect to
chlorophyll a.
Figure 2, represent a biostabilizing system in which
the chlorophyll a don’t decompose to phaeophytene and
only a little increase of chlorophyll b is observed when
the aged time increasing indicated by an increase in the
fluorescent intensity with a wav e length shift of the band
located at 726 nm to 713 nm.
In Figure 3, we show the sample with a high extract
leave concentratio n. In this case a pigment aggregation is
present and then a red shift of maximum fluorescent,
respect to 675 nm obtained for the samples with 4 g and
8 g per 20 ml of extract leave concentration. The maxi-
mum fluorescence is located at 686 nm. The band located
at 724 nm present a higher intensity compared with sam-
ples of Figures 1 and 2, corresponding to low extract
leave concentration.
When the water to TEOS molar ratio is reduced the
biostabilization is breaking as we can observe in Figure
4, where the fluorescent spectra for an extract leave con-
centration of 8 g / 20 ml and water to TEOS molar ratio
of 5 is shown.
The fluorescent contribution between 630 nm to 750
nm corresponds to chlorophyll b with bands located at
668 nm and 710 nm characteristic of such chlorophyll
specie. The aging effect produce a rapid denaturation of
chlorophyll compound and a blue shift occur. The typical
red fluorescence disappear and a blue-shifted band at
about 580 nm is present [14], indicating the decomposi-
tion of the photosyste m II. This situation is also ob served
in samples of ortho-amnino-substituted porphyrin,
H2T(o-NH2)PP, embedded in SiO2 matrix [15].
The large shift is typical for weakening of chlorophyll
– protein interactions in pigment – protein complex
(PPCs). The carotenoid radical cation formation play one
important role in photosynthetic pigment-protein com-
plexes, and quench chlorophyll fluorescence [18,19]. The
chlorophyll molecule contain the phytyl group associated
to carotenoid radical, the low water content retarding the
gel formation and the denaturation of photosystem II
active the carotenoid function of protect the photosyn-
thetic apparatus by quenching chlorophyll triplet state
which inhibits the formation of singlet state oxygen
[20-22], and stabilize light-harvesting protein structures
[23-25]. The active presence of carotenoid radicals is
indicated by the blue shift band located at 583 nm [26].
The difference in structure due to concentration of ex-
tract leave and water to TEOS molar ratio, are reflected
on the fluorescence differences discussed previously. In
Figure 5 we observe that, among the differences in rela-
tive intensities for bands characteristic of chlorophyll a
and b, related with the photosystems I and II, we observe
that the position of maximum fluorescence of the band
corresponding to PSII at about 680 nm, is red shifted
conform the concentration is increased. This result is in
agreement with results reported by Furukawa et al., for
layered silica/surfactant mesostructured this films con-
taining chlorophyllous pigments where the red shift in
optical measurements indicates the aggregation of pig-
ments [9].
The fluorescent bands, for samples labeled by C1 and
C2 concentrations in Figure 5 are very similar, with a
lighter red shift for sample C2 respect to C1. However
for aged samples these bands present higher differences
indicating that a higher biostabilisation is obtained for
concentration C2, as we can observe in Figure 6.
550 600 650 700 750
Normalized fluorescence int ensity
Em ission wavelen g th (nm)
R5, C2
C3
C1
C2
Figure 5. Fluorescent emission spectra of spinach leaves,
with concentration of 4 g / 20 ml (C1), 8 g / 20 ml (C2), 50 g
/ 20 ml (C3) and 8 g / 20 ml with water to TEOS molar ratio
of 5 (R5,C2), embedded in silica xerogel matrix measured
after gelling.

Effect of Aging on Chlorophyll Species Embedded in Silica Xerogels Matrix11
550 600650 700750
Normalized fluorescence intensity
Emission wavelength (nm)
R5, C2
C2
C1
C3
Figure 6. Fluorescent emission spectra of spinach leaves,
with concentration of 4 g / 20 ml (C1), 8 g / 20 ml (C2), 50 g
/ 20 ml (C3) and 8g / 20ml with water to TEOS molar ratio
of 5 (R5,C2), embedded in silica xerogel matrix measured
after 36 months of gelling.
4. Conclusions
The PSII fluorescence comportment for spinach extract
of leaves embedded in silica xerogel matrix, give us in-
formation about the microstructural evolution during the
silica-gel-glass conversion.
We obtain a higher PSII stability for extract of leaves,
with concentration of 8 g / 20 ml and water to TEOS
molar ratio of 11.67 embedded in xerogel matrix, which
remain bioactive over a very long period of time,
whereas for a water to TEOS molar ratio of 5 a blue-shift
fluorescence is observed indicating the PSII denaturizing
and formation of fluorescing aggregates associated with
the activation of carotenoids radical s.
In samples with a water to TEOS molar ratio of R = 11,
and concentration between 4 g to 50 g per 20 ml the
chlorophylls molecules entrapped in silica xerogel re-
main bioactive ov er a long period of time.
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