Effect of Aging in the Structure of Silica Xerogels with Incorporation of Extract’s Leaves

doi:10.4236/njgc.2011.11002

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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)

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.

Email: [email protected]

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

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

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

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

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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