Surface-enhanced Raman and fluorescence joint analysis of soil ...

analytica chimica acta 616 (2008) 69–77
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Surface-enhanced Raman and fluorescence
joint analysis of soil humic acids
G. Corrado b , S. Sanchez-Cortes a,∗ , O. Francioso b , J.V. Garcia-Ramos a
a Instituto de Estructura de la Materia, CSIC, Serrano 121, 28006 Madrid, Spain
b Dipartimento di Scienze e Tecnologie Agroambientali, Università degli Studi di Bologna, V.le Fanin 40, Bologna 40126, Italy
article
info
abstract
Article history:
Received 20 November 2007
Received in revised form
1 April 2008
Accepted 8 April 2008
Published on line 15 April 2008
Keywords:
Surface-emhanced Raman
Scattering
Surface-enhanced Fluorescence
Humic acids
Spectroscopic imaging
Surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) combined
emissions were used in this work to the analysis of humic acids (HA). This study
examined HA structure at different pH and HA concentrations and assessed the structural
differences taking place in HA as a result of various amendment trials. Raman and fluorescence
emissions behave in opposite ways due to the effect of the metal surface on
the aromatic groups responsible for these emissions. The information afforded by these
techniques can be successfully employed in the structural and dynamic analysis of these
important macromolecules. The surface-enhanced emission (SEE) spectra, that is the sum
of the Raman and the fluorescence emissions, were acquired by using both macro- and microexperimental
configurations in order to apply imaging and confocal Raman and fluorescence
spectroscopy techniques on the analysis of HA.
© 2008 Published by Elsevier B.V.
1. Introduction
Humic substances (HS) are the most important organic components
of soil and the most widely distributed organic
products of biosynthesis on the face of the earth [1]. In addition
to the quantitative importance of HS, these substances act as
a sink and source of C, they influence the soil fertility through
the release of nutrients and affect the fate of contaminants
leading to soil remediation [2,3]. Although HS are widespread
in soils and water, during a long time their structure remained
unknown, but in the last years the knowledge on these compounds
has notably increased [2,4–6]. HS are divided into two
main groups: humic acids (HA) and fulvic acids (FA) each displaying
a different solubility in acidic media [7].
The inherent complexity of HS has seriously limited
the application of more traditional optical spectroscopies,
such as Raman and fluorescence spectroscopy, in structural
and dynamic studies of HS. Fluorescence spectroscopy has
been used so far in the characterisation of HA by several
authors [8–11]. However, the high complexity of HS limits the
application of this technique to these compounds. Raman
spectroscopy is a technique, which has been widely applied,
in structural studies of organic molecules and biological compounds
[12]. However, normal Raman spectroscopy cannot
be employed in the structural characterisation of HS due to
the intense fluorescence emission, which overlaps the Raman
bands.
Over recent years, several optical spectroscopy techniques
(Raman, IR and fluorescence) have undergone a renaissance
due to the notable characteristics of metal nanostructures
(MNs) where localised surface plasmon (LSP) can occur. LSP
leads to a remarkable local electromagnetic field enhancement
owing to the high absorption of light in the vicinity of
metal nanoparticles and thus induces a huge enhancement of
∗ Corresponding author. Tel.: +34 915616800; fax: +34 915645557.
E-mail address: imts158@iem.cfmac.csic.es (S. Sanchez-Cortes).
0003-2670/$ – see front matter © 2008 Published by Elsevier B.V.
doi:10.1016/j.aca.2008.04.019

70 analytica chimica acta 616 (2008) 69–77
spectroscopic signals, mainly in the case of emission signals
of molecules placed in the proximities of the surface [13–15].
Raman scattering is greatly enhanced as a consequence
of LSP. For this reason, the surface-enhanced Raman scattering
(SERS) technique can be successfully applied to the
study of highly fluorescent molecules in water, due to
the fluorescence quenching occurring on the metal surface
via a charge-transfer mechanism [16]. This effect
enables Raman to be applied to the characterisation of HS
[17–20].
SERS spectra can provide important information on the
HS structure in function of factors such as the humification
degree [21], extraction and purification processes [22], the soil
amendment trials [23], HS extraction fractions [17,24] and pH
[25]. The growing importance of SERS in the analysis of HS
has recently been highlighted in several reviews dealing with
techniques applied to the analysis of HS [26–28]. SERS has also
been used to examine pesticide–HA interaction [29]. However,
SERS is not a quantitative technique because of effects such
as the resonance Raman and the adsorption on surfaces [30],
although these characteristics can also serve to study structural
and dynamic processes undertaken by macromolecules
in the presence of surfaces [31].
The surface-enhanced fluorescence (SEF) technique has
not been employed as much as SERS because of chargetransfer
effects, which take place on the surface and which,
in turn, lead to an actual quenching of the fluorescence. SEF
effect is also induced by LPR near a metal surface [32,33].However,
the net effect seems to vary depending on the distance
between the fluorophore and the surface, as well as on the
intrinsic quantum yield of the fluorophore [34]. In general,
the fluorescence is not quenched if the molecule is relatively
far from the surface. The estimated optimal distance for a
good SEF enhancement is achieved above 100 Å [35,36]. At distances
further than this value, SERS and SEF signals can be
simultaneously emitted and registered for molecular species
placed in the vicinity of MNs [36–38]. Since the average size
of HA is larger than this value [39], the fluorophores included
in the HA structure could be good candidates to give rise to
intense SEF + SERS joint emission spectra, without the intrinsic
quenching observed on a surface. This is possible due to the
fact that the HS cross-sections of either Raman or fluorescence
are of the same order on MNs, and thus, a surface-enhanced
emission (SEE) is seen for HS adsorbed on these substrates
(Fig. 1, bottom). Furthermore, the SEF emission affords information
on the HA macromolecules adsorbed onto a surface,
Fig. 1 – Top: experimental set-up for SEE measurements. Bottom left: a SEM image of typical Ag nanoaggregates. Bottom
right: scheme displaying the adsorption of HA micelles and the emission of Raman a fluorescence combined process.

analytica chimica acta 616 (2008) 69–77 71
i.e. on the aggregation process undergone by these macromolecules,
which are of importance to understand how these
compounds are attached on the inorganic matrices existing
in soils. This fact represented an evident advantage regarding
the normal fluorescence spectra obtained in solution.
Spectroscopic imaging and micro-confocal techniques
have been developed for both Raman and fluorescence spectroscopy
with promising analytical applications [40], and
could also be of interest in the study of HA, as demonstrated by
previous studies [41]. The combination of surface-enhanced
techniques and microscopy is also a powerful methodology
which has attracted much attention in the analysis of biological
systems such as cells or bacteria [42,43] and which could
be very helpful in the characterisation of HS.
In this work we analysed SEE spectra of HA. This work
represents the first time that SEF technique is used in the characterisation
of HA. The HA compounds selected for this study
were extracted from a soil amended over a period of 30 years
under several conditions and the corresponding control experiments
in order to evaluate the structural variations induced
by the different treatments. The SEE spectra were acquired by
using both macro- and micro-experimental configurations in
order to investigate the abilities of the new imaging and confocal
Raman and fluorescence spectroscopy techniques on the
analysis of these HA.
2. Materials and methods
The soil samples analysed (horizon Ap, 0–40 cm depth) were
taken from plots of a 30 plus year field experiment conducted
at the agricultural farm of the University of Bologna’s Agricultural
Faculty (Cadriano, Italy). This soil is classified (Soil Survey
Staff, USDA SCS 1989) as a Typic Udochrept.
The experimental design included plots amended over a
30-year period with cattle manure (CM) and crop residues (CR)
constituted by wheat straw or corn stalks (each biomass followed
in succession), with unamended soil as control (C). CM
was added to the plots at the following rate: 6 t ha −1 of dry matter
after wheat tillage and 7.5 t ha −1 of dry matter after corn
tillage. All the plots examined, including C, were fertilised with
100 kg N ha −1 yr −1 added in the form of ammonium nitrate.
Each treatment (CM, CR and C) was conducted in triplicate
using a randomized block design.
Analyses were carried out on soil samples taken from:
(i) unamended soil in 1972 (C 0 ), and (ii) after 30 years (C 30 )
and (iii) after a 30-year amendment period with CM and CR.
The extraction procedures used to isolate these samples have
already been described in Ref. [23].
to enhance the electromagnetic field on the metal surface. A
typical Ag MNs used to record the SEE spectra is shown in Fig. 1
(bottom).
Samples for SEE measurements were made by adding an
aliquot of an aqueous HS solution, prepared by solving 1 mg of
the corresponding humic fraction in 1 mL of tri-distilled water,
to 1 mL of the silver colloid until the desired concentration.
The pH of the mixture was adjusted by adding 0.1 M NaOH or
HNO 3 . All solutions were prepared with tri-distilled water.
The SERS spectra were recorded by means of a Renishaw
Raman Microscope System RM2000, using the macroconfiguration
equipped with a notch filter and an electrically
refrigerated CCD camera (575× pixels) as indicated in Fig. 1.
The Raman and fluorescence emission spectrum is recorded
by the CCD camera. The 514.5 nm line of an Ar + laser was used.
The laser power at the sample was 2.0 mW. Spectral resolution
was 2 cm −1 . Micro-SERS spectra were obtained by using a 100×
objective (NA = 0.9). Confocal micro-SERS and Raman images
were recorded by using a PRIOR motorised stage coupled to the
microscope with a 1 m step. The SERS spectra were baselined
to withdraw the contribution of the fluorescence by using the
algorithm provided by the Origin 6.0 Program.
3. Results and discussion
3.1. SERS spectra of humic acids
Fig. 2 shows the SERS spectra of HA at pH 10.0, after correction
of the fluorescence background. At pH 10.0 the SERS spectra
of all HA samples are dominated by two very strong and narrow
bands, at 1308 and 1611 cm −1 , which can be attributed
to the aromatic part of HS and in particular to polycyclic aromatic
hydrocarbon (PAH) structures. The nature of these PAH
residues is still unknown, but the similarity of the SERS pattern
observed at high pH and the Raman spectra of, phenanthrene,
perylene [45] and coronene [46] compounds suggests that similar
structures could exist in HA, either from anthropogenic
2.1. SERS spectroscopy
Colloidal silver nanoparticles were prepared by using hydroxylamine
hydrochloride as reducing agent. In a recent work, a
deep study of Ag MNs prepared by reduction with hydroxylamine
(AgHx) was accomplished [44]. AgHx was activated by
the addition of potassium nitrate, which induced the particle
aggregation to form other larger aggregations. This process
is necessary when using these systems as a substrate for
SERS, since specific morphological requirements are needed
Fig. 2 – SERS spectra of the different HA samples
(10 gmL −1 ) employed in this work after correcting the
fluorescence background to better show the Raman
features.

72 analytica chimica acta 616 (2008) 69–77
or natural origins [2,7]. The situation seems to be different
in the case of FA, since no PAH bands are observed in these
compounds [24]. The presence of PAH in the SERS spectrum
recorded at alkaline pH, suggests that PAH residues might be
mainly localised inside the HA structure, since a HA structure
expansion is expected at high pH due to ionisation of
carboxylic and phenolic groups [47]. Although the macromolecular
expansion is limited by chemical cross-linkages
[48], it seems to be enough to allow a higher exposure of the
polyaromatic groups to the metal surface. The bands corresponding
to PAH residues are resonantly enhanced as revealed
the appearance of the overtone and combination bands at
1948, 2577, 2913 and 3228 cm −1 in the SERS spectra (Fig. 2).
However, we cannot deduce that PAH groups are quantitatively
predominant in the HA structure, since these groups
undergone a selective enhancement by SERS.
The SERS spectra in Fig. 2 were normalised to the water
band appearing at 3420 cm −1 . As can be seen, the CR and CM
samples display a lower SERS intensity in comparison to the
control samples C 0 and C 30 . In order to better understand this
behaviour we carried out a more detailed SERS study of HA
at different pH values using the C 30 sample (Fig. 3), since this
sample was the one, which displayed the most intense variation.
The bands at 1308 and 1611 cm −1 observed at pH 10.0
(Fig. 3a) decreased markedly at pH 5 (Fig. 3d), as a consequence
of: (i) an increase of intermolecular interactions, due to both
a decrease of electric repulsions and an increase [47] of H-
bonds and (ii) coagulation of smaller HA micelles into larger
ones [49]. These facts lead to a macromolecular shrinkage.
Below this pH value, a significant enhancement of the SERS
intensity was observed (Fig. 3e), which is related to the HA
adsorption tendency increase of at low pH as also reported by
several authors [50,51]. The HA adsorption increase is related
to the higher hydrophobic nature of HA at low pH caused
by the negative charge decrease upon protonation of acidic
HA groups. The increase of HA molecules adsorbed on the
metal is further intensified due to the reduction of the area
per molecule induced by the molecular shrinkage occurring
at low pH [52].
The SERS spectrum seen at low pH changes with respect to
that observed at high pH (Fig. 3a). The analysis of the Raman
broad bands seen at low pH is rather difficult owed to the sum
of different components and the possible contribution from
carbonaceous materials such as charcoal [53,54], which may
be incorporated in the humic structure of that can be formed
due to photodegradation as indicated by other authors [55].
Nevertheless, the different Raman profile with respect to charcoal
Raman spectrum [54], the variation of this spectrum with
the pH, as revealed below, and the appearance of bands which
are attributable to functional groups actually existing in HA,
such as benzoic, polyphenolic and aliphatic ones, discharge
the possibility of a massive photodegradation of the adsorbed
HA as an effect of light irradiation.
The dependency of SERS intensity on pH is better seen in
the inset figure of Fig. 3, which displays a minimum at ca.
5.5. Recently, Terashima et al. [52] have demonstrated that
the apparent pK (pK app ) of HA is approximately 5.5 and, as
a consequence, at pH 5.5 drastic changes take place in several
physical properties, such as surface tension, surface area
occupied by HA molecules when adsorbed onto a surface
and HA hydrophobicity. A similar pH value was reported by
other authors when studying the aggregation tendency of HA
[56,57].
It therefore follows that the lower SERS intensity of CR and
CM samples in comparison to the control C 0 and C 30 , can
be attributed to the effect of amendants. The introduction of
lignin in the HA structure may increase the stability of the
macromolecule due to the higher H-bonds formed between
OH groups afforded by lignin. In the case of CM, the enrichment
in fatty acids, due to the treatment with manure, also
lead to a higher stabilisation of the macromolecule due to
the formation of hydrophobic interactions between aliphatic
chains of fatty acid residues.
3.2. Surface-enhanced emission spectra of humic acids
Fig. 3 – SERS spectra of C 30 sample (10 gmL −1 ) after
fluorescence correction at the following pH: (a) 10.0, (b) 8.0,
(c) 6.0, (d) 5.5 and (e) 3.0. Inset: variation of the 1609 cm −1
SERS band on changing the pH.
Fig. 4 compares the normal Raman spectrum of the C 30 sample
(1 gmL −1 ) with SEE spectrum of the same sample in the
presence of Ag NPs. The emission spectrum of the solution
shows the Raman bands of water at 1640 cm −1 (very weak) and
3420 cm −1 (strong) and a broad fluorescence band produced
by HA centered at 630 nm, while no Raman band of HA are
seen. In contrast, the SEE spectrum of HA was characterised
by strong Raman bands in the 1200–1650 cm −1 region, and by
a fluorescence emission with a maximum at 650 nm, which
is threefold intensified in relation to that seen in aqueous
solution. The intensification of Raman bands corresponding
to the HA and the shift observed for the fluorescence emission
maximum suggests structural changes in the HA upon
adsorption on the metal. Thus, the SEE spectrum of the C 30
sample provided a combined enhanced Raman and fluorescence
information because the cross-sections corresponding

analytica chimica acta 616 (2008) 69–77 73
Fig. 4 – Surface-enhanced emission (SEE) spectra of C 30
sample (1 gmL −1 ) in absence (a) and presence (b) of Ag
nanoparticles.
Fig. 6 – Panel (a): variation with pH of the surface-enhanced
fluorescence emission (excitation at 514.5 nm and emission
at 625 nm). Panel (b): the surface-enhanced Raman
scattering intensity of 1611 cm −1 band (excitation at
514.5 nm) measured from the C 30 , CR and CM samples at a
HA concentration of 10.0 gmL −1 .
to both emissions are of the same magnitude order. Although
the total emission measured from adsorbates adsorbed on
metal NPs is also a combined luminescence signal with contributions
from both the molecule and the metal background
luminescence [58], we can consider that the latter one is negligible
in relation to the luminescence of HA in the SEE spectra.
SEE spectra recorded at three different HA concentrations
are shown in Fig. 5. At high concentration, the fluorescence
signal predominates over the Raman signal, while at lower
concentrations the opposite behaviour is witnessed. This is
probably due to the formation of HA multilayers on the Ag
NPs, which leads to higher fluorescence intensities as the multilayer
width is increased due to a lower quenching effect by
the metal. The predominance of the SERS signal over the SEF
Fig. 5 – Surface-enhanced emission (SEE) spectra of C 30
sample on Ag nanoparticles at the following
concentrations: (a) 10.0 gmL −1 , (b) 1.0 gmL −1 and (c)
0.1 gmL −1 .
one (in fact limit of detection of 1–100 ppb can be obtained,
depending on the case) points out the powerful analytical possibilities
of SERS in comparison to the fluorescence, in spite of
the higher sensibility of fluorescence over the Raman effect.
The effect of pH on the Raman and fluorescence of HA samples
is shown in Fig. 6. The Raman intensity changes with the
pH reaching a minimum at pH 5.5 (Fig. 6b). In contrast, the
analysis of the fluorescence emission revealed an opposite
behaviour, i.e. a maximum at precisely pH 5.5 (Fig. 6a).
This contrary behaviour of the fluorescence and SERS signals
of HA can be explained on the basis of the structural
modifications induced on HA as a consequence of the acidic
groups ionisation on increasing the pH. Other authors have
also reported similar variations with pH of the HS fluorescence
emission [8,59]. The HA uncoiling or structural expansion
occurring in the pH range between 8 and 10 provoked an opposite
response on the fluorescence and Raman intensities. By
one hand, it favours the approach of the inner PAH residues
of HA to the metal leading to an enhancement of the SERS
signal. On the other hand, this expansion induces the fluorescence
quenching due to the higher exposure of fluorophores
to the aqueous polar medium and the charge-transfer induced
by the closer metal surface.
In contrast, at pH 5.5 the HA is compacted, and consequently
the PAHs residues remain inside the HA structure, and
therefore, far from the metal surface, yielding a weaker SERS
intensity. However, the fluorescence signal is enhanced due
to the lower fluorescence quenching of fluorophore groups,
which are therefore protected from the aqueous medium
and the metal surface quenching effects. This behaviour
is similar to the fluorescence change in proteins due to
folding–unfolding processes observed on changing the pH [60].
At pH below 5.0, the Raman intensity is remarkably intensified
due to the higher adsorption tendency of HA, as
mentioned above, but the fluorescence quenching observed
at lower pH is now attributed to the protonation of its acidic

74 analytica chimica acta 616 (2008) 69–77
Fig. 8 – (a) Intensity of HA 1611 cm −1 Raman band
measured for the C 30 sample adsorbed on the Ag
nanoaggregates which were selected and labelled with
letters in the top optical micrograph (b). Excitation at
514.5 nm.
ate behaviour between CR and C 30 samples due to the presence
of a high amount of aliphatic chains introduced by the residual
fatty acids of manures, which may contribute to a further
stabilisation of the HS structure through hydrophobic interactions,
as also corroborated in a previous work [63].
3.3. Micro-SEE spectra of HA
Fig. 7 – Spectroscopic images obtained by mapping the
intensity of the Raman 1611 cm −1 band (excitation at
514.5 nm) (b) and the fluorescence emission (excitation at
514.5 nm and emission at 590 nm) (c) from the C 30 sample
adsorbed on the Ag nanoaggregates shown in the optical
image (a).
groups and the lower quantum efficiency of protonated groups
[8].
Although this is a general behaviour of HA, as we have seen
for many HA types, they also display differences, which can be
attributed to the structural changes induced by amendants,
which the soil have received for over 30 years. For instance,
the CR and C 30 samples show the minimum and maximum
variations of fluorescence and Raman signals, respectively.
This can be attributed again to the presence of a larger number
of phenolic groups in CR sample due to the incorporation
of lignin derivatives coming from the residual crops used in
the amendment. Several authors have also detected a higher
sensitivity to pH in HS bearing a higher content in phenolic
hydroxyl groups [8,61,62]. CM sample exhibits an intermedi-
Micro-SEE spectra were obtained from Ag nanoaggregates
immobilised on a glass substrate corresponding to the optical
image shown in Fig. 7a. One of the distinct advantages of
micro-Raman instruments is the possibility to record images
and maps, which enable the distribution of a certain species to
be followed by recording the intensity of a characteristic band.
The intense band at 1611 cm −1 was used as reference to study
the SERS intensity obtained from different points in the same
metal aggregate onto which the C 30 was adsorbed. Fig. 7b and
c shows the images obtained by mapping the intensity of the
1611 cm −1 Raman band and the fluorescence at 572 nm (with
excitation at 514.5 nm) in 50 m × 50 m areas of the aggregate
shown in the optical image of Fig. 7a. The spectroscopic
images indicate that both the Raman and fluorescence signals
are greatly enhanced in the vicinity of the metal aggregate,
thus highlighting the fact that the emission spectra are actually
produced by surface-enhanced Raman and fluorescence
effects with a negligible contribution from the bulk.
The maps of Fig. 7b and c indicate that the emission intensity
is higher in the lower part of the nanoaggregate. According
to the electromagnetic mechanism of LSP-based surfaceenhanced
spectroscopy [13–15], the enhancement depends on
the morphology of metallic clusters. In addition, the existence

analytica chimica acta 616 (2008) 69–77 75
observed features was interesting to gain an insight into the
adsorption of a macromolecular heterogeneous system such
as HA.
Fig. 8a indicates that the Raman intensity depends on the
morphology of the aggregate studied. The “d” nanoaggregate
was the most efficient at enhancing the Raman intensity.
This can be attributed to the more appropriate morphology
for electromagnetic field enhancement on the surface of this
aggregate. In contrast, aggregate a yields a very low Raman
intensity, even if the size is comparable to that of the “d”
aggregate. This study affords interesting information to investigate
the influence of the Ag nanoaggregate morphology on
the SERS enhancement.
Fig. 9 shows that the spectral pattern undergoes a slight
variability from point to point, which was not observed in
the corresponding macro-SEE spectra. This effect can be
attributed to the intrinsic structural heterogeneity of HA,
which can be adsorbed onto the surface through the different
chemical groups existing in their structure.
It is worth noting that the different features detected in
the micro-SERS spectra obtained at the different points indicated
in Fig. 8a precisely matched the bands resulting from
the curve-fitting of the macro-SERS spectrum registered in
macro-conditions for the same sample, as indicated in the
upper spectrum of Fig. 9 (see the arrows). This fact clearly
indicates that in the surface-enhanced analysis of macromolecular
systems the spectrum obtained in macro is actually
the sum of the different spectra, which can be obtained, in
micro-conditions.
4. Conclusions
Fig. 9 – Bottom: micro-SERS spectra of the C 30 sample
(excitation at 514.5 nm) measured at the different Ag
nanoaggregates selected in the optical image shown in
Fig. 8b. Top: curve fitted SERS spectrum recorded for the
same sample in macro-conditions.
of areas with large SERS enhancements, so-called hot spots,
has also been reported on metallic NPs. Suitable hot spots are
believed to be formed, for example, at a junction between
two metallic nanoparticles [64,65] and are highly localised.
Thus it seems that the local morphology of the lower aggregate
of Fig. 7a is highly favourable to an enhancement of the
electromagnetic field, as deduced from the high SEE signal
observed. This is probably due to the existence there of hot
spots.
A random Raman mapping was also performed in a region
where different Ag nanoaggregates having different sizes and
shapes can be found (Fig. 8b). The micro-SERS spectra recorded
for the points labelled with letters in Fig. 8b are represented
in Fig. 9, while the Raman intensity of the 1611 cm −1 band
is plotted in Fig. 8a for all the examined points. The analysis
of the overall Raman intensity and relative intensity of the
SEE is a promising combined Raman plus fluorescence technique
to be applied in the structural and dynamic analysis of
HA. SEE signal is very sensitive to the HA shrinkage, which
in turn may vary with the pH, the concentration and the
structural modifications of HA induced by different soil treatments.
Raman and fluorescence emissions follow an opposite
behaviour due to the effect of the metal surface on the groups
responsible for these emissions. This fact can be related to
the different shrinking state of the HA structure, which may
vary with the pH or the amendment trials. At high HA concentration
the SEE spectra are dominated by the fluorescence
emission. However, at low HA concentrations the SERS signal
predominates over the fluorescence and a limit of detection
as low as 1 ppb can be achieved.
The sensitivity of SEE is further increased in microspectroscopy.
A very low detection limit can be achieved in
micro-conditions, with the detection of few HA molecules
(probably much lower taking into account that HA molecules
placed in hot spots are those contributing to a highest extent
to the overall SEE signal). The micro-SERS spectra show a high
variability from point to point due to the inherent complexity
of HA micelles.
The SEE technique has promising applications in the influence
of other factors affecting the HA structure such as: origin,
humification rank, and interaction with pollutants. Indeed,
this is a work that we are currently doing.

76 analytica chimica acta 616 (2008) 69–77
Acknowledgements
The authors wish to acknowledge grants from the Dirección
General de Investigación, Ministerio de Educación y Ciencia (Project
FIS2004-00108, FIS2007-63065) and Comunidad Autonoma de
Madrid (Project MICROSERES-S-0505/TIC-0191). GC acknowledges
a grant from the University of Bologna. This research
was also supported with funds provided by (i) University
of Bologna (ex quota 60%, 2006). The authors would like
to thank Profs. Giovanni Toderi and Guido Baldoni and
Dr. Gianni Giordani for kindly providing the historical soil
samples.
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