Hot Topic Tomrj Contents

The Open Magnetic Resonance Journal
Volume 3, 2010

ISSN: 1874-7698

HOT TOPIC:

Applications and New Developments of Magnetic Resonance
Techniques in Soil Science

Guest editors:
Pellegrino Conte Universit degli Studi di Palermo
Anne E. Berns Forschungszentrum Jlich
Andreas Pohlmeier Forschungszentrum Jlich
Giuseppe Alonzo Universit degli Studi di Palermo
Contents

Editorial 14

Proton Nuclear Magnetic Resonance (NMR) Relaxometry in Soil 15
Science Applications
Julia V. Bayer, Fabian Jaeger and Gabriele E. Schaumann

Proton NMR Relaxometry as a Useful Tool to Evaluate Swelling 27
Processes in Peat Soils
Fabian Jaeger, Anastasia Shchegolikhina, Henk Van As and Gabriele Ellen Schaumann

Investigation of Iron(III)-Release in the Pore Water of Natural Sands 46
by NMR Relaxometry
Ivonne Mitreiter, Sascha E. Oswald and Frank Stallmach

A Possible Difference in the Surface Relaxivity of Costal and Inland 52
Sands
Joseph P. Hornak, Gianni Ferrante, Andrew Coy and Evan R. McCarney

Relaxation in a Natural Soil: Comparison of Relaxometric Imaging, 57
T1 T2 Correlation and Fast-Field Cycling NMR
S. Haber-Pohlmeier, S. Stapf, D. van Dusschoten and A. Pohlmeier

Low-Field NMR of Water in Model Soils 63
Oscar Sucre, Federico Casanova, Andreas Pohlmeier and Bernhard Bluemich

MRI in Soils: Determination of Water Content Changes Due to Root 69
Water Uptake by Means of a Multi-Slice-Multi-Echo Sequence (MSME)
A. Pohlmeier, F. Vergeldt, E. Gerkema, H. Van As, D. Van Dusschoten and H. Vereecken

Effect of RF Field Inhomogeneity and Sample Restriction on Spectral 75
Resolution of CP/MAS-
13
C NMR Spectra of Natural Organic Matter
Anne E. Berns and Pellegrino Conte

Interaction of a Recombinant Prion Protein with Organo-Mineral 84
Complexes as Assessed by FT-IR and CPMAS
13
C NMR Analysis
Fabio Russo, Liliana Gianfreda, Pellegrino Conte and Maria A. Rao

CPMAS
13
C NMR Characterization of Leaves and Litters from the 89
Reafforestated Area of Mustigarufi in Sicily (Italy)
Pellegrino Conte, Claudio De Pasquale, Etelvino H. Novotny, Gianluca Caponetto,
Vito Armando Laudicina, Maurizio Ciofalo, Michele Panno, Eristanna Palazzolo,
Luigi Badalucco and Giuseppe Alonzo

13
C-NMR Chemical Shift Databases as a Quick Tool to Evaluate 96
Structural Models of Humic Substances
Christian Nyrop Albers and Poul Erik Hansen

14 The Open Magnetic Resonance Journal, 2010, Volume 3 Editorial
Open Access
EDITORIAL
Applications and New Developments of Magnetic Resonance Techniques in Soil Science
In soil and environmental sciences, magnetic resonance techniques are nowadays used for a large variety of applications
such as those related to the characterization of natural organic matter, to the analysis of the interactions of pollutants with or-
ganic and inorganic moieties in environmental compartments, and to the evaluation of fluid flow in porous media. Recent ad-
vances in electronics allowed not only the development of NMR on solids and semi-solid samples, but also the evolution of in
situ NMR equipments. In addition to classical NMR applications, magnetic resonance imaging (MRI) of soil-water and tracer
transport processes in soil (e.g. spatial and temporal changes of soil-water distributions or the movement of wetting fronts, root
growth and water uptake) and NMR relaxometry and diffusometry for pore space exploration in natural porous media are now
important fields in soil studies.
This special issue includes the conference proceedings of the session SSS23 titled Applications and new developments of
magnetic resonance techniques in soil science which was held in April 2009 in Vienna (Austria) within the Soil System Sci-
ences program of the European Geoscience Union (EGU) General Assembly (http://meetingorganizer.copernicus.org/
EGU2009/session/947). During this first session in the EGU Assemblies dealing with NMR, all the aspects of NMR spectros-
copy were accounted for: from the limits of the classical NMR techniques such as CPMAS
13
C NMR spectroscopy applied to
soils and humic substances, to the developments of innovative NMR techniques for soil science investigations such as low field
and relaxometry NMR.
The paper selection encompassed in the present special issue of The Open Magnetic Resonance Journal reflects the diver-
sity of magnetic resonance applications in the field of soil and environmental sciences. It does of course not claim completeness
in these fields, but is meant to give an interesting and hopefully inspiring excerpt of these continuously expanding research
fields. The first part of the issue presents new developments and applications of low field NMR and relaxometry, followed by a
paper on magnetic resonance imaging applied to the analysis of plant-soil interactions. The last part of the special issue con-
cludes with the classical high field solid and liquid state NMR spectroscopy. Namely, a paper on the evaluation of the limits of
CPMAS
13
C NMR spectroscopy due to imperfect field homogenization along the coil is presented together with a second paper,
where interactions among a prion protein and a soil-like system were investigated. Finally, the last paper deals with the sugges-
tion of a quick method for validity evaluation of the humic substances models which were suggested in the literature.

Pellegrino Conte
Universit degli Studi di Palermo
Dipartimento di Ingegneria e Tecnologie Agro-Forestali
v.le delle Scienze 13, edificio 4
90128, Palermo
Italy
E-mail: pellegrino.conte@unipa.it
Anne E. Berns
Institute of Chemistry and Dynamics of the Geosphere
ICG-4: Agrosphere
Forschungszentrum Jlich GmbH
52425 Jlich
Germany
E-mail: a.berns@fz-juelich.de

Andreas Pohlmeier
Institute of Chemistry and Dynamics of the Geosphere
ICG-4: Agrosphere
Forschungszentrum Jlich GmbH
52425 Jlich
Germany
E-mail: a.pohlmeier@fz-juelich.de

Giuseppe Alonzo
Universit degli Studi di Palermo
Dipartimento di Ingegneria e Tecnologie Agro-Forestali
v.le delle Scienze 13, edificio 4
90128, Palermo
Italy
E-mail: alonzo@unipa.it

Conte et al.; Licensee Bentham Open.

This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the
work is properly cited.
The Open Magnetic Resonance Journal, 2010, 3, 15-26 15

1874-7698/10 2010 Bentham Open
Open Access
Proton Nuclear Magnetic Resonance (NMR) Relaxometry in Soil Science
Applications
Julia V. Bayer*, Fabian Jaeger and Gabriele E. Schaumann
Department of Environmental and Soil Chemistry, Institute of Environmental Sciences, University of Koblenz-Landau,
Fortstrae 7, 76829 Landau, Germany
Abstract: Proton NMR relaxometry is a very powerful tool for investigating porous media and their interaction with wa-
ter or other liquids and the mobility and interaction of organic molecules in solution. It is commonly used in material sci-
ence or earth science. However, it is only scarcely applied in soil science although it shows great potential for helping to
understand water uptake into the soil matrix and processes occurring at the solid-liquid interface at soil particle surfaces.
This review introduces proton NMR relaxometry in the context of soil science and discusses the most important applica-
tions of the method in this field. Relevant results from different applications of NMR relaxometry in soils are described
and research gaps identified. Some original data is presented concerning biofilm formation in soils, which was investi-
gated using proton NMR relaxometry. NMR relaxometry is a sensitive, informative and promising method to study pore
size distribution in soils as well as many kinds of soil physicochemical processes, among which are wetting, swelling or
changes in macromolecular status. It is further a very helpful method to study interactions between molecules in soil or-
ganic matter and can serve to study the state of binding of water or organic chemicals to soil organic matter. Relaxation
times determined by NMR relaxometry are sensitive to various factors that play a role in soil-water interaction which is
both an advantage and shortcoming of the method: NMR relaxometry can be applied to numerous investigations in soil
science, but at the same time interpretation of the results may be very difficult in such complex and heterogeneous sys-
tems like soils.
Keywords: NMR relaxometry, soil, porous media, water, swelling, wetting.
INTRODUCTION
Proton NMR relaxometry is commonly used in geo-
sciences, e.g. in oil exploration, and material sciences [1, 2].
It is a powerful tool for non-destructive investigations of
pore size distributions of porous media, water content, water
uptake and re-distribution as well as molecule mobility and
non-covalent binding mechanisms. The technique can be
adapted for use in soil investigations, but so far has only
been used sparsely. More frequently used for structural
analysis is NMR spectroscopy which is able to give insight
into the molecular structure of soil organic matter (SOM). It
has been used extensively for the determination of humic
acid (HA) and fulvic acid (FA) structures and other organic
constituents in SOM in either liquid or solid state. Detailed
reviews of the state of the art of NMR spectroscopy in natu-
ral organic matter NOM, heterogeneous material and poly-
mers are given elsewhere [3-5]. More specific details of
NMR spectroscopy applications in soil, SOM and biological
systems, including several other NMR techniques like mag-
netic resonance imaging (MRI) and the investigation of mo-
bility of deuterated or fluorinated compounds, can be found
e.g. in [6-11].
This review focuses mainly on the application of proton
NMR relaxometry in soil science, especially
1
H NMR

*Address correspondence to this author at the Department of Environmental
and Soil Chemistry, Institute of Environmental Sciences, University of
Koblenz-Landau, Fortstrae 7, 76829 Landau, Germany; Tel: +49 (0)6341-
280-565; Fax: +49 (0)6341-280-576; E-mail: bayer@uni-landau.de
relaxometry, including some relevant studies on other porous
media and magnetic resonance imaging (MRI) studies of
soils. Unfortunately, NMR relaxometry as used for studies in
geosciences cannot be transferred one to one for studying
soils, as e.g. the pore system differs from that found in rock
formations. The main challenge for application to soil is, in
this context, its huge complexity and heterogeneity and the
up to now only scarcely understood soil organic matter [12,
13]. Nevertheless, efforts have been made to use NMR re-
laxometry to describe pore size distributions in soils, as well
as processes occurring during water uptake, i.e. wetting,
swelling of organic matter and re-distribution of water.
Apart from pure NMR relaxometry, MRI studies, based
on the same measurement principle as NMR relaxometry,
may help to get insight into soil water interactions as they
provide spatial resolution additional to the temporal resolu-
tion. Many studies employ MRI for understanding water
uptake into soils or similar porous media and gain qualitative
and quantitative information about local water distribution:
Theoretical considerations about formation of preferential
flow pathways have been confirmed by MRI; water and hy-
drocarbon distribution and displacement have been evaluated
and water distribution within the pore system can be ob-
served, e.g. [14-22]. However, the resolution of MRI is much
lower than that of NMR relaxometry and no detailed infor-
mation about the pore size distribution or water properties
within the pores can be determined from such measurements.
Apart from
1
H NMR relaxometry, other nuclei such as
13
C or
19
F can be used for relaxation studies of liquids in porous
media giving insight into distribution of those liquids [23,
16 The Open Magnetic Resonance Journal, 2010, Volume 3 Bayer et al.
24] and the pore size distribution of the porous media, e.g.
[25, 26].
Gaining insight into water distribution in soils is espe-
cially important for nutrient and contaminant distribution,
which is of interest for agricultural applications and in rela-
tion to aquifer contamination [27]. Any substance entering
the soil pore system interacts with the solid surface and,
therefore, depends on solution distribution and interaction
with the matrix. Water uptake in soils is not a simple distri-
bution problem as e.g. preferential pathways form due to the
existence of macropores and different surface wettabilities of
the solid surface influence the wetting process [28]. Also,
water does not only enter pores, but interacts with the or-
ganic matter coatings and organic colloids present in the
pore system. This changes the solid surface and, hence, the
pore system itself. Model calculations are often inadequate
in describing water uptake into and the interaction of water
with the soil matrix [27, 28]. NMR relaxometry, therefore,
offers a great potential for investigating soil water interac-
tions without the need for modelling or sample destruction.
The method can be used in-situ, especially with more recent
developments in mobile NMR techniques that could be used
directly in the field [29-37].
Addressing both soil scientists interested in the use of
these techniques for their own purpose and NMR specialists
providing new promising NMR relaxometry tools which
help to obtain further insights into soil processes, the objec-
tive of this contribution is to outline and discuss fields of
application of this technique in soil science. Although, dif-
ferent NMR methods are commonly used in soil science ap-
plications, this review focuses mainly on proton NMR re-
laxometry, due to the complexity of the field.
BASICS OF NMR RELAXOMETRY
This section is addressed mainly to the reader unfamiliar
with the field of NMR. Many atomic nuclei posses a non-
zero spin and an intrinsic magnetic moment parallel or anti-
parallel to the spin. The spin is associated with a non-zero
magnetic moment () via the relation = J, where is the
gyromagnetic ratio and J the spin angular momentum. is
constant, but assumes different values for different nuclei.
When placed in an outer magnetic field B (conventionally
along the z-axis), the spins orientate and precess about the
external field with the Larmor frequency, which is character-
istic for each nucleus and dependent on the strength of the
outer magnetic field (e.g. hydrogen nucleus 42.6 MHz at 1
Tesla): f= B/2 (e.g., [1]).
A radio frequency (RF) pulse with the characteristic
Larmor frequency is applied and the spins are flipped into an
angle to the external magnetic field (B
0
) causing a magneti-
sation (M
0
). In most applications the RF pulse turns the spins
in 90 or 180 direction (in a certain pulse sequence). After
the RF pulse is switched off the spins relax to their equilib-
rium orientation and the apparent magnetisation induced by
the RF pulse decays. The measured signal is called the free
induction decay (FID) [2]. The relaxation process generally
is a first order process. It is characterized by the relaxation
time, which is the reciprocal of the relaxation rate constant.
Two different relaxation mechanisms are involved in the
magnetisation decay, which are the longitudinal or spin-
lattice and transverse or spin-spin relaxation [2].
The spin-lattice relaxation time T
1
depends mainly on the
interaction of the spins with their environment often referred
to as the lattice, hence the name. T
1
describes how effective
interactions between the spin system and the environment
are in exchanging magnetic energy. If strong interactions
between the spin system and the environment lead to a fast
exchange of energy, the equilibrium state is reached fast and
T1 is short. Measuring T
1
can be very time consuming and is,
so far, not often used in soil science applications, although it
may be the more appropriate measure than T
2
in many cases
[1, 2].
The spin-spin relaxation time T
2
normally refers to the
relaxation due to variable molecular interactions or diffusion
in the slightly inhomogeneous magnetic field. The transver-
sal relaxation process is not based on energy exchange, but
originates from a dephasing of the precessing spins, e.g., due
to slight differences in Larmor frequency due to local field
inhomogeneity [2]. Variations in the magnetic field caused
by neighbouring nuclei are stronger in solids than in liquids
where spins can move freely and inhomogeneities due to
neighbouring spins are small. As the dephasing of the spins
can only take place in the presence of a longitudinal mag-
netisation T
2
can be smaller than or equal to T
1
, but it can
never be longer [1].
While bulk liquids lacking additional means of interac-
tion reveal long proton relaxation times in the range of sec-
onds, limitation of mobility can reduce T
2
. Contrary to T
2
, T
1

can be either increased or reduced by a reduction in mobility,
depending on the Larmor frequency and the correlation time
for the relaxation-relevant interaction [2]. Molecular diffu-
sion in field gradients affects T
2
but not T
1
, because no en-
ergy exchange is involved in this relaxation mechanism [38].
The relaxation rate due to diffusion in field gradients is pro-
portional to the diffusion coefficient and the square of local
field gradients [2]. The local field gradients increase with
increasing external field strength. Therefore, measurements
in systems like soils, where local field gradients are the rule,
are to be carried out preferentially in low fields up to 10-50
MHz. Field cycling NMR explicitly investigates the field
and frequency dependency of T
1
and T
2
at proton Larmor
frequencies between 10 kHz and 40 MHz or higher and is,
therefore, a promising tool to study dynamic molecular in-
teractions and to distinguish between the molecular effects
and effects of local field gradients or sample heterogeneity
[39]. In traditional high-resolution NMR spectroscopy,
where Larmor frequencies are generally above 250 MHz,
large T
1
/T
2
ratios are the rule.
Relaxation Times T
1
and T
2
in Porous Systems
With T
1
and T
2
of protons of bulk water in the range of 1-
3 seconds, bulk relaxation processes are very slow. If con-
fined in porous media, relaxation is often controlled by solid-
fluid-interactions at the surfaces of the pore space. Water
molecules diffuse and eventually reach a pore wall surface
where there is a finite probability that their spins are relaxed
due to interactions with fixed spins, paramagnetic ions or
paramagnetic crystal defects. Further transversal relaxation
occurs via diffusion in local field gradients. The total relaxa-
Proton Nuclear Magnetic Resonance (NMR) Relaxometry The Open Magnetic Resonance Journal, 2010, Volume 3 17
tion rate is, therefore, the sum of bulk relaxation (B) and
surface relaxation (S) and, for T
2
, of relaxation due to diffu-
sion in field gradients [1]:
FG diff S B total
S B total
T T T T
T T T
2 2 2 2
1 1 1
1 1 1 1
1 1 1
(1)
The surface relaxation term contains information of the
pore system and is, therefore, further analysed. Relaxation
time at the surface is determined by the residence time of the
spin at the surface. The longer the residence time the higher
the probability for interaction with the surface and, therefore,
relaxation. As long as this surface relaxation is slower than
the transport of unrelaxed spins to the surface the fast-
diffusion or surface-limited regime [40] is fulfilled. Water
molecules can transit the pore several times before being
relaxed and the magnetization decay in an individual pore is,
therefore, spatially uniform and depends on the surface-to-
volume ratio. Surface relaxation is then related to the internal
surface area S, internal pore volume V and the surface relax-
ivity [1] which is strongly influenced by paramagnetic ions
on the surface like Mn
2+
or Fe
3+
:
surface-limited:
r V
S
T
S

2 , 1 2 , 1
2 , 1
1
= (2)
where r is the pore radius and is the shape factor (1, 2, 3
for planar, cylindrical and spherical pore geometry) [41].
If, in contrast, the magnetic decay is controlled by the
transport of the molecules to the surface the conditions of the
slow-diffusion or diffusion-limited regime [40] are met. This
may be the case if pores are large or surface relaxation is
strong, e.g., due to the presence of effective paramagnetic
centres.
diffusion-limited:
2
2 1
1 1
r
c
D
T T
S S
= = (3)
where D is the diffusion coefficient and c is a shape-
dependent factor. Note that in the case of diffusion limitation
T
1S
and T
2S
are equal. Relaxation times in the diffusion-
limited regime depend on temperature in the same way as the
diffusion coefficient. In this case, relaxation times are not
spatially uniform, which results in a multiexponential mag-
netic decay, even within a single pore, and relaxation is addi-
tionally dependent on pore shape [1]:
Converting NMR Signals into Relaxation Time Distribu-
tions
For data analysis in NMR relaxometry several different
algorithms and software is applied. These are, e.g., the soft-
ware WinDXP from Resonance Instruments (UK) or Con-
tin used by Bruker (USA) [42], which are device specific, or
UPEN, a software developed at the University of Bologna
[43, 44]. Depending on the chosen program and parameters,
the analyses may lead to differently strongly separated peaks
in the relaxation distribution. One such parameter is the
weight factor used in WinDXP to account for different signal
to noise ratios. An example is given in Fig. (1) for a water
repellent soil sample using a weight factor of 0.1 (Fig. 1a)
and 20 (Fig. 1b). The differences in peak separation and
peak number clearly demonstrate the importance to consider
the effect of evaluation parameters on the resulting relaxa-
tion time distribution [45]. Samples can only be compared to
each other on the basis of comparable evaluation parameters
using the same software.
WATER IN SOILS AND PEATS
Water uptake and redistribution in soils as well as inter-
action of water with SOM is of great importance for, e.g.,
contaminant sorption or nutrient availability. The availability
of water for plants itself is important in agricultural sciences
and is determined by the water content, the matric potential
() and the water retention curve of a soil. Hereby, a matric
potential of -1.5 MPa is defined as the permanent wilting
point where plant growths is limited due to water shortage; a
matric potential of -0.03 MPa is defined as the field capacity
which is the amount of water held in a soil after excess water
was drained due to gravitation [28]. Measurement of water
retention curves by standard soil science methods is time
consuming and cannot be carried out in situ.
A first effort to use low-field NMR relaxometry for
analysis of water in soils was made by Prebble and Currie
already in 1970 by measuring T
1
(at 2.7 MHz) [46]. They
used several sands, soils and a vermiculite as sample mate-
rial and added different amounts of water. Three states of
water in soils were identified: i) At very low water contents
water was tightly bound to the clay or sand interface, but no
relationship with plant unavailable water was found ( = -1.5
MPa); ii) with increasing water content the water seemed to
be independent of the clay lattice and water content calcula-
tions resulted in values close to the real amount of water
added and iii) further addition of water lead to an incomplete
relaxation during the measurement time indicating the pres-
ence of bulk water ( = -0.03 MPa). The presence of various
states of water was confirmed for peat samples by McBrierty
et al. [47]. In a detailed study using high field NMR re-
laxometry (300 MHz), differential scanning calorimetry
(DSC) and thermogravimetric analysis (TGA) the binding of
water in peat was investigated and up to four different water
states were found, with two forms of loosely bound water,
bulk water and tightly bound water that did not freeze at
temperatures down to 160 K. The loosely bound water froze
around 210 K and bulk water at 273 K, indicating also the
temperature range above which each water form became
mobile. Drying and re-watering of peat samples did shrink
and swell the peat matrix and with that changed the amount
of loosely bound water, but the amount of strongly surface
associated water was similar after each change in moisture
status. Non-freezing water was associated with hydration
water, i.e. water in a gel like layer at the solid surface or wa-
ter that chemically interacts with the hydrophilic moieties on
the surface [47]. Therefore, the amount of non-freezing wa-
ter could be an indicator for surface properties. The interac-
tions of water molecules at the surface of particles is respon-
sible for the thickness of the bound water layer or phy-
sisorbed water, which can be up to 3 molecular layers. The
relaxation rate increases (linearly) with the amount of solid
surface present, as shown for water clay suspensions, due to
the relaxivity offered by the solid surface [48].
The relaxation mechanisms at the solid liquid interface
are manifold and paramagnetic substances have an important
influence. The coverage of only 0.01% with Fe(III) of a sil-
ica surface was enough to increase surface relaxivity by an
order of magnitude [49]. However, Mn(II) seems to be an
even stronger relaxing agent than Fe(III) with a relaxation
acceleration effect of up to three times stronger in solutions
[50, 51]. The effect of paramagnetic ions on the surface re-
laxivity seems to be restricted to one atomic layer at the sur-
face of a particle as shown for Mn(II) on calcite particles
[50]. Further increase in manganese concentrations in calcite
water systems did not increase surface relaxivity further and
also Mn(II) inside calcite particles did not contribute to sur-
face relaxivity either [50]. The effect of paramagnetic sub-
stances on the relaxation rate was observed to be much
stronger when they are adsorbed to the solid surface, due to
the restricted molecular motion of the adsorbed species
which in turn results in a longer rotational correlation time
for the coordinated water molecules. Nevertheless, bulk re-
laxation is also accelerated in the presence of dissolved par-
amagnetic ions [49-51]. The relaxation acceleration effect of
paramagnetic substances in the bulk solution is dependent on
the speciation of the ion [49, 51]. It was suggested that the
relaxation acceleration decreases from hexa-aqua complexes
to aqua complexes with a reduced number of exchangeable
protons to organic-complexed ions to dispersed colloids.
Therefore, the acceleration of the bulk relaxation rate in
comparison to pure water may give additional information
on the ion environment in complex soil solutions [51].
Due to the dependency of the relaxation times on the wa-
ter binding and distribution NMR relaxometry can be used to
describe the water environment: water in small pores or
bound water relaxes faster than that in large pores or free
water, due to increased accessibility of the solid surface.
Gaining information about water uptake and redistribution in
soil systems is of high importance, e.g., for agricultural sys-
tems or prediction of contaminant distribution. Several stud-
ies so far have been carried out investigating water uptake
into soils or clays using relaxation time distributions deter-
mined by
1
H-NMR relaxometry [45, 47, 52-59]. Relaxation
time distributions generally showed three or four separate
peaks representing different water states or water in different
pore systems. The boundary conditions vary between publi-
cations, but as a general rule one can differentiate between
micropores or tightly surface bound water at small relaxation
times (e.g. T
2
: below 60 ms, sometimes separated into sev-
eral peaks), mesopores or loosely bound water at medium
relaxation times (e.g. T
2
: 60 300 ms) and macropores or
bulk water at long relaxation times (e.g. T
2
> 300) [47, 52,
60]. Some researchers found several relaxation time peaks at
medium relaxation times. The loosely bound water relaxing
with these relaxation times was possibly associated with dif-
ferent separate environments that did not allow water ex-
change at time scales of the relaxation measurements. Over
the course of water uptake relaxation time distributions
shifted towards smaller relaxation times and peaks at shorter
relaxation times increased in size (Fig. 1a and b) [45, 53-55,
60].
The shift of relaxation times towards shorter times indi-
cates water movement into smaller pores, which is contrary
to the common model of water imbibition into porous media
with hydrophilic pore walls where small pores are filled first
due to capillary forces. As an explanation it was suggested
that pore walls become increasingly hydrophilic with in-
creasing soil-water contact time [45, 58] or that micropores
that initially collapsed upon drying were reformed during
water uptake by formation of water-swollen gels [53]. The
latter process was referred to as swelling [53]. However, the
definition of swelling is not used consistently in other
publications.
Generally, the water uptake and re-distribution were
found to be separated into fast and slow processes which can
last up to weeks. The activation energies calculated for the
fast and slow processes by Todoruk et al. [53] indicate that
they are fundamentally different: The fast process had acti-
vation energies of ~ 42kJ mol
-1
which is in the upper range
of diffusion associated processes. The slow component,
however, had activation energies of > 80kJ mol
-1
indicating
chemical transformations like ester hydrolysis or more com-
plex rearrangements of SOM components [53]. During wet-
ting of a soil the hydrogen bonds of SOM components and
mineral surface, which had been formed previously during
drying, have to be broken apart in order to restore hydro-
philic surface conditions; this process would be slow and

Fig. (1). Comparison of T2 relaxation time distribution of a water repellent sample directly after water addition and 19 days later using two
smoothing values: a) weight factor 0.1 and b) weight factor 20. Data taken from [45] and adjusted.
energetically unfavourable, leading to high activation ener-
gies [53]. Other authors distinguished more clearly between
wetting and swelling as two separate more or less independ-
ent processes [54]. Wetting was suggested to be considerably
faster (indicated by a shorter time constant of relaxation time
changes) than swelling in hydrophilic soils and primarily
associated with the properties of the solid surface (whether
mineral or organic). In order to wet a surface it needs to be
hydrophilic, however, prolonged contact of water with an
organic hydrophobic soil particle surface could render it wet-
table and, hence, would allow further processes like swelling
to take place. This is displayed in NMR measurements as a
slow change in relaxation times towards shorter relaxation
times. Swelling here was defined as the hydration of SOM
which increases the thickness of the SOM coating or SOM
particle. This in turn would lead to a decrease in interparticu-
lar pore size [45, 54]. The process of swelling may be of
high relevance for contaminant fixation in soils as it may
influence interactions of contaminants with SOM by e.g.
increasing the available sorption site, forming new sorption
domains or changing its rigidity [54, 61].
As described above it is necessary to have a hydrophilic
surface in order to enable instant wetting. However, in sys-
tems like soil, surfaces change when in contact with water
and originally hydrophobic surfaces become wettable after
prolonged contact with water. The breakdown of a hydro-
phobic surface during wetting is thought to be fast in com-
parison to swelling. It was suggested to exploit that fact and
use low-field
1
H-NMR relaxometry for soil wettability de-
terminations [56, 57, 62]. In order for a liquid in porous me-
dia to be relaxed efficiently it needs to be in contact with the
solid surface. Theoretical considerations suggest that relaxa-
tion times of hydrophobic samples are longer than that of
wettable samples enabling a better proton exchange [56, 57].
T
2
of water repellent soil samples and model systems was
found to be larger than 1000 ms, but that of wettable samples
~100 ms [56, 57]. As described above water repellency of
organic coatings on particle surfaces normally breaks down
after contact with water, therefore, relaxation times of water
repellent and wettable sample should eventually reach the
same equilibrium. The decrease of relaxation time and ap-
proach of a similar equilibrium was confirmed in two studies
and the time for reaching the equilibrium was dependent on
the sample [56, 57].
Proton NMR relaxometry studies of water in soil systems
allow to distinguish processes taking place during water up-
take. It is also possible to differentiate between water in sev-
eral environments, i.e., bound, loosely bound and free bulk
water. Furthermore, influences of factors like paramagnetic
substances in solution and on the solid surface have been
characterised and partly quantified. However, it is still nec-
essary to quantitatively describe the processes occurring dur-
ing water uptake into soils, such as wetting and swelling and
evaluate their environmental impact like their involvement in
nutrient or contaminant distribution.
PORE SIZE DISTRIBUTION IN SOILS
It is well established that porosity and pores size distribu-
tions can be derived from relaxation time distribution of geo-
logical formations, like rocks, sandstones or permafrost and
gas hydrate sediments, or materials such as ceramics (e.g. [2,
63-65]). However, even in rocks comparison of pore size
distributions from different samples has to be considered
carefully. The iron concentrations in rock formations are
probably high enough to ensure constant surface relaxivity
(compare section water and porous media), nevertheless,
shifts in relaxation time distributions may not only be due to
differences in pore size distributions, but differences in the
amount of paramagnetic substances present in the sample
[41, 49]. The presence of paramagnetic substances on a
coated silica gel reduced the relaxation time of water close to
the surface so much that the monomodal relaxation time dis-
tributions were changed to bimodal distributions, thereby
identifying microporosity of the surface [49].With increasing
SOM content the number of identified water compartments
increased from three to four suggesting a correlation between
pore system and organic matter [52]. An even more detailed
relationship between soil components and pore sizes was
identified in another study: The relaxation time of soil sam-
ples was found to be dependent on sand, silt, clay and SOM
content, but the degree of correlation was dependent on the
pore system, i.e. micro-or mesopores. The transverse relaxa-
tion times of micropores correlate with clay and SOM con-
tents, but those of mesopores with silt, sand and SOM [66].
In another study the influence of kaolinite addition to
sandy samples was investigated [67] and found an increase
in relaxation rate with increasing amount of kaolinite present
in the sample. This was ascribed to the increasing surface
area (increase in smaller pores) and the higher surface relax-
ivity of kaolinite (one reason for this higher surface relaxiv-
ity may be the presence of iron in the octahedral layers of
kaolinite). However, at a certain amount of kaolinite the re-
laxation rate increased less. This was assumed to be the point
where all sand surfaces were covered in kaolinite and the
surface relaxivity was stable, leaving only decreasing pore
size and changing pore geometry responsible for changes in
T
1
.
A slightly different approach to determine pore mobile
and immobile fractions in a wetland soil was used by Culli-
gan et al. [68]: The sample (a sphagnum peat moss) was
saturated with water and T
1
was determined (at 122 MHz),
then a 1 mM Gd
3+
solution was added and T
1
was deter-
mined again. As the Gd
3+
solution was added under condi-
tions where diffusion is negligible, this second measurement
sampled only the mobile pore space. It was found that 43%
of the pore space showed a fast relaxation time (T
1
= 35 ms),
and 56% exhibited a longer relaxation time of T
1
= 165 ms.
The first was assumed to represent the pore space filled by
Gd
3+
solution, whereas the latter only by water, therefore,
confirming the existence of two porosities in the wetland
peat.
One main assumption when converting relaxation time
distribution into pore size distributions is that pores are not
interconnected or more specific relaxation starts and ends
within one pore. This may apply to geological formations
which have larger pores than soils, but does not hold true for
soils. Also, the pore drainage in soils can be considered to
not necessarily be total, i.e. some pores drain while others
retain their water [60]. Further assumptions are that the sur-
face relaxation is constant throughout the pore system and
the shape factor of the pores is constant and known [55]. In
most studies assessing pore size distributions the fast diffu-
sion regime is assumed, so that relaxation time is influenced
only by the surface relaxivity of the solid surface and the
relaxation time of the bulk phase [66]. The surface relaxivity
can be determined from volume to surface area ratios which
in turn can be determined from e.g. nitrogen adsorption or
mercury porosity measurements [55, 63, 66].
The application of NMR relaxometry to determine soil
pore size distributions so far has been mainly qualitative.
Several studies agree that relaxation time distributions of soil
samples are related to pore sizes, but do not directly and
quantitatively describe pore size distributions [53, 69]. The
study conducted by Hinedi et al. (1993) was probably the
first one to derive a real pore size distribution from a relaxa-
tion time distribution, but did not verify the outcomes by
comparing them to results from conventionally obtained pore
size distributions [55]. A qualitative comparison of NMR
derived and conventional determined pore size distributions
was undertaken in two later studies, but NMR relaxometry
was recommended only as an additional method to conven-
tional pore size determination to characterize pore connec-
tivity [60]. However, a quantitative comparison between
pore size distributions derived from NMR and conventional
methods so far has been mainly conducted for several rock
types [63]. Pore sizes, determined by NMR relaxation meas-
urements in comparison to mercury porosimetry, were over-
estimated by an order of magnitude. Mercury porosimetry is
based on the Washburn equation (
x
r
v
l

2
cos
= , where v is
the rate of liquid entry into the capillary, r is the capillary
radius,
l
is the liquid surface tension, is the viscosity of the
liquid, x is the distance penetrated, and is the contact angle
[70]). It, therefore, tends to reflect more pore throats than
pore sizes, leading to an underestimation of the real pore size
[63]. Just recently the application of NMR relaxometry (T
2

measurements at 2 MHz) for determination of pore size dis-
tributions by quantitatively comparing it to conventional
pore size distributions derived from water retention curves
was verified for several soil types [66]. In this new approach,
the relaxation time pore size relation revealed two separate
regions. The condition for the fast-diffusion regime [40] was
fulfilled for T
2
< 10 ms. For larger T
2
values, a transition
from the fast-diffusion to the intermediate-diffusion regime
[40] for finer textured soil samples, and transition from the
intermediate-diffusion to the slow-diffusion regime [40] for
sandy soil samples was determined. Additionally, the true
bulk relaxation time was used instead of the hypothetical one
of free water commonly assumed for such investigations
[66].Consequently, proton relaxation in larger pores was
governed by surface relaxivity and self diffusion of water.
However, for simplification, the condition for the fast-
diffusion regime was assumed as fulfilled for all pore sizes in
this study. A good consistency (R
2
= 0.98) between pore size
distributions determined by conventional soil water retention
measurements and
1
H NMR relaxometry was found using
the two different surface relaxivities for micro-and
mesopores (for details on calculations see [66]).

As described above, the determination of conventional
soul water retention curves is still necessary in order to be
able to calculate surface relaxivities. In order to use the
whole time-saving potential of the NMR measurements an
independent method for the determination of surface relaxiv-
ities is necessary. Additionally, changes in pore sizes during
water uptake as often reported have to be investigated further
as they may not only be attributed to swelling of organic
matter on particle surfaces or water re-distribution into pores
previously not available, but also to the formation of new
pore systems due to microbial activity.
COMPLEXATION OF PARAMAGNETIC IONS IN
SOIL SOLUTIONS
Both relaxation times are greatly reduced in the presence
of paramagnetic ions. The strength of the effect depends on
the ion environment and specification. The interaction of
paramagnetic ions with FA or HA in solution, thus, can be
investigated using
1
H NMR relaxometry. Variations between
Mn(II), Cu(II) and Fe(III) relaxation times suggested that
different complexation mechanisms were at work in several
studies [51, 71-73]: No change or only minimal change was
found for solutions containing sulphosalicylic acid and
Mn(II) in contrast to solutions with only Mn(II), suggesting
the formation of outer sphere complexes, as the rotational
motion of the ions was not affected [71]. However, Cu(II)
and Fe(III) solutions were strongly affected by the presence
of sulphosalicyclic acid (reduction of relaxation time with
increasing concentration of sulphosalicyclic acid) suggesting
the formation of inner sphere complexes [71]. Contrary find-
ings were reported by Melton et al. for solutions of Lauren-
tian HA [72]: Relaxation times of solutions with Cu(II) de-
creased only slightly with increasing concentration of HA
[72]. The formation of stable or labile metal complexes,
therefore, seems to be very dependent on the organic mate-
rial. Interactions of organic compounds and FA or HA in
solution were also investigated by changing concentrations
and environmental parameters in the solution and their ef-
fects on relaxation times were observed. A difference in the
interaction of HA and monoaromatic compounds was found
depending on the aromaticity and also very strongly on pH
[73]. Relaxation acceleration due to interaction with dis-
solved and colloidal Fe and Mn species in soil solutions
causes a wide range of relaxation times in dependence of the
Fe and Mn speciation [51].
FA and HA were shown to form - complexes with hy-
drophobic organic compounds like dichlorophenol. FA was
less effective in forming such complexes than HA which was
attributed to the stronger hydrophobic character of HA [74].
Two NMR relaxometry studies using
13
C-labeled acenaph-
tone and fluoro-acenaphtone both found evidence that the
mode of interaction of FA and acenaphtone depends strongly
on the concentration of FA in solution and the solution pH
[75, 76].
Investigations of such interactions may help to under-
stand the fate of organic compounds in the aquatic environ-
ment and are partly transferable to soil systems; however, the
soil matrix is so much more complex and exhibits so much
more opportunity for interactions apart from the soil solu-
tion, that a direct transfer is not possible.

MOBILITY AND NON-COVALENT BINDING
MECHANISMS OF ORGANIC MOLECULES IN THE
SOLID ORGANIC MATTER
NMR relaxometry can be used to probe the spin envi-
ronment and, therefore, gain information about binding and
association forms of the molecule under investigation. These
investigations are indirectly related to soils as they can help
predicting behaviour of organic compounds in the environ-
ment. Main constituents of SOM are fulvic acids (FA) and
humic acids (HA) and several studies investigated the inter-
action and non-covalent binding forms of organic molecules
or metal ions with HA and FA (e.g. [71, 73-79]). The identi-
fication of rigid and flexible structures within organic mate-
rials is also possible [79, 80]. The reported temperature de-
pendence of rigid and flexible domains within HA correlated
well with glass transition temperature determined by several
other authors using differential scanning calorimetry (e.g.
[81, 82]). Another recent study [61] reported a correlation of
decrease in matrix rigidity of a peat sample with an increase
of proton relaxation time (T
2
). After heating a sample in an
airtight container its matrix rigidity was reduced and relaxa-
tion time increased, indicating a higher mobility of the or-
ganic matter involved. After two weeks proton relaxation
time had decreased to the original value and matrix rigidity
increased. Suggested by earlier studies obtained from de-
tailed DSC and TGA analysis [83-85], it was assumed that
this may be due to the formation of cross-links between or-
ganic molecules via water molecules (physicochemical ma-
trix aging). The thermal and moisture history is expected to
be linked closely to the mobility of organics and the matrix
rigidity [61]. As rigid and flexible domains probably show
different sorption towards contaminants the identification
and quantification of such domains within SOM is of interest
for modelling contaminant sorption behaviour.
MICROBIAL INFLUENCES
Microorganisms can form extended networks, so called
biofilms, in order to relieve water stress and use nutrients
more efficiently. These biofilms are formed of extended ex-
tracellular polymeric substances (EPS) networks which bind
water very effectively and form highly hydrated gels [86].
Biofilms or small biofilm-like structural units can also be
formed in soils.
The change of the spin environment within such biofilms
compared to bulk water was tested by NMR relaxation or
MRI [9, 87, 88]. In aqueous solutions the monomodal relaxa-
tion time distribution (T
2
at 85 MHz) of water became bi-
modal in the presence of a biofilms. However, in a porous
model system of glass beads the resolution of the peaks was
not possible due to the relaxation effects of the solid surface
of the pore system. MR images of the same samples con-
firmed biofilm distribution true to the optical examination
[87] proving the applicability of the methods for such sys-
tems.
In soil samples the detection of biofilm growth is not that
easy and bacteria do not form free biofilm inside pores, but
use EPS to attach themselves to the particle surface and en-
hance transport of nutrients [86]. Microorganisms in soils are
mainly attached to particle surfaces and primarily found in
pores with diameters of 1-30 m [89, 90].
Enhancing microorganism activity in soil samples re-
sulted in a stronger shift of the relaxation time (T
2
) towards
shorter T
2
in treated (enhanced microbial activity) than un-
treated samples over the course of water uptake. This could
be due to the increased production of EPS in the treated
samples which may have reduced sizes of existing pores or
formed a new micropore system. However, the contributions
of other processes in reducing relaxation times like swelling
of SOM or the change of surface relaxivity due to bacterial
growths could not be excluded up to now [88] and further
research in this area is needed.
Effects of Biofilm on Proton Relaxation Time Distribu-
tions in Model Soil Systems
In a qualitative study, the effects of bacterial biofilm on
transverse relaxation time distribution of water in biofilm
reactors, used as model soil systems, at 2 MHz (Maran 2,
Resonance Instruments, UK) were investigated (not pub-
lished). Special designed glass bottles (height x diameter: 12
cm x 5 cm; volume: 160 cm) with two bottle closures (at the
top and bottom) were filled with glass beads of different par-
ticle sizes or with natural soil (sandy soil, sieve fraction 63
m to 2 mm). Some of the reactors were inoculated with a
biofilm producing isolate (99% sequence identity with Si-
norhizobium sp. TB8-10-II, isolated from a waste water sand
filter) and relaxation time distributions were measured after
incubation time of 5 to 8 days. Optical inspection of the glass
bead reactors showed biofilm growth after this time; for the
soil reactor a similar growth was assumed. Fig. (2) shows the
setup of the reactor system (left hand side) and a sketch of a
filled reactor (right hand side). Reactors were filled with up
to five layers of glass beads with particle sizes ranging from
5 mm to 150 m (decreasing particle sizes from glass closure
to bottle middle) to prevent particle outflow. Layer D in Fig.
(2) represents the domain studied in the NMR relaxometer
(i.e. filled with the different growing materials). 2.5 L of a
30 g L
-1
Trypticase
TM
Soy Broth solution (BD Diagnostic
Systems, Heidelberg, Germany) was used as a culture me-
dium and was pumped with 8 mL h
-1
into to a dropping fun-
nel to prevent contamination (Fig. 2). A second pump (circu-
late pump with 900 mL h
-1
) was responsible for the flow of
culture medium through the reactor. After finishing the ex-
periment, the reactors were dried using a pump. However,
this was only possible for the 3 mm glass beads as the pres-
sure was not high enough to dry the other size fractions.
1
H NMR measurements were performed using a CPMG
pulse sequence [91]. The number of 180 pulses ranged be-
tween 8192 (soil) and 14336 (3 mm glass beads) with con-
stant number of scans of 256. Echo spacing ranged between
150 s (soil) and 300 s (glass beads). The objective was to
achieve a signal to noise ratio between 50 and 100. The repe-
tition time was set individually for every reactor and chosen
based on three to six times the longest T
2
and was 3-10 s.
Relaxation time distributions were calculated from the decay
curves with the WinDXP software (Resonance Instruments,
UK) running a zeroth order regularisation to perform a con-
tinuous distribution of exponentials applying the BRD (But-
ler, Reeds and Dawson) algorithm [92]. The relaxation time
distributions consisted of 128 time constants with associated
amplitudes. The time constant range was 1-10000 ms, and
the weight factor for the regularization was 0.5 for all
biofilm reactors.

Fig. (2). Setup of the reactor system (left hand side) and a sketch of a biofilm reactor filled with glass beads (right hand side).
Transverse relaxation time distribution of water in reac-
tors filled with 3 mm glass beads (Fig. 3) or 500-350 m
glass beads (Fig. 4) consisted of two to three peaks. Peak 0
(3 mm glass beads: T
2
= 40-90 ms; 500-350 m glass beads:
T
2
= 30-40 ms) may be a fitting artefact, because its exis-
tence and position was not reproducible in the replicate sam-
ples. Furthermore, its intensity was in the range background
noise. Position of Peak 1 was T
2
= 300 ms for 3 mm glass
beads without biofilm and up to 500 ms for 3 mm glass
beads with biofilm (Fig. 3). For 500-350 m glass beads,
Peak 1 was determined around T
2
= 150 ms for reactors with
and without biofilm (Fig. 4). This suggests that Peak 1 repre-
sents water between the contact areas of the glass particles,
because its position decreased and its intensity increased
with decreasing particle size in the reactors without biofilm.
In the time scale of the NMR experiment, this water is not
exchanging with water in larger pores as represented by Peak
2, and, thus, may be represented by an individual peak. In
the inoculated glass bead reactors, it may include water in-
side the biofilm matrix, because its intensity tended to in-
crease with increasing biofilm dry mass. This suggestion is
supported by the finding for agar gels, which were used as
model biofilm, were T
2
ranged between 1100 ms and 100 ms
for agar concentrations of 1.0-10 g L
-1
(not shown). Peak 2
may represent water in large antiparticle pores, because its
position decreased with decreasing particle size (3 mm glass
beads: T
2
= 2300 ms without and ~ 2000 ms with biofilm;
500-350 m glass beads: T
2
= 1100 ms without and ~ 800
ms with biofilm).

Fig. (4). Transverse relaxation time distribution of water in reactors
filled with 500-350 m glass beads with and without fresh biofilm.

For both types of glass beads, biofilm growth resulted in
a shifting of peak 2 towards smaller T
2
values (Fig. 3, 4).
This suggests decreasing interparticle pore diameters and/or
changes of the surface relaxivity caused by biofilm on the
glass bead surfaces. Additionally, the intensity of peak 1
tended to increase in the reactors with fresh biofilm. This
observation was not determined for the rewetted biofilm
(Fig. 3), suggesting structural changes due to drying.

filled with 3 mm glass beads with and without fresh biofilm, and
with rewetted biofilm after air drying of the reactor with fresh
biofilm.
10 100 1000
0
3
6
9
Peak 0
Peak 2 3 mm glass beads
without biofilm
fresh biofilm
rewetted biofilm
P
r
o
p
o
r
t
i
o
n

o
f

H
2
O
t
o
t
a
l

[
%
]
T
2
[ms]
Peak 1
10 100 1000
0
3
6
9
500-350 m glass beads
without biofilm
fresh biofilm
P
r
o
p
o
r
t
i
o
n

o
f

H
2
O
t
o
t
a
l

[
%
]
T
2
[ms]
Peak 0 Peak 1
Peak 2
The T
2
distribution of water in reactors filled with natural
soil (sieve fraction 63-2000 m) consisted of three peaks,
representing water in different pore types (Fig. 5). Bacterial
inoculation resulted in considerable changes of the T
2
distri-
bution. The intensity of peak 1 increased and peak 2 and 3
showed a trend to decreasing intensity. This suggests that
pore diameters of larger inter-particular pores decreased and
that the amount of smaller pores increased due to the forma-
tion of biofilm inside the soil matrix. One inoculated soil
filled reactor was found to be clogged and also changes in
the T
2
distribution were very strongly developed (biofilm 2
in Fig. 5), suggesting a very strong biofilm growth. A clog-
ging due to small soil particle sizes can most likely be ex-
cluded, because particles smaller 63 m were removed by
sieving prior to the experiment. Furthermore, the control
reactor without inoculation showed no evidence of clogging.
Biofouling and pore clogging is also known in technical ap-
plications like tubes, membrane filters and sand filters [93].
The results of the soil filled reactors are qualitatively compa-
rable to those of Jaeger et al. and support their assumption
that biofilm formation affected the T
2
distribution of water in
soil samples with higher microbial respiratory activity [88].

filled with natural soil (sieve fraction 63-2000 m) with and with-
out fresh biofilm.

A 50 times higher transverse than longitudinal relaxivity
was determined for agar gels at 30 MHz. This finding can be
interpreted in terms of a reduced rotational mobility of the
water molecules due to water structuring of the polymer
[94]. Thus, a combination of T
1
and T
2
measurements can be
suggested for a more detailed study of biofilm or other gel
phases, e.g. inside the SOM matrix [54]. This may be helpful
to determine different water states and to discriminate be-
tween the effects of water mobility and pore size distribution
in biofilm or gel containing porous media.
OUTLOOK
The potential of proton NMR relaxometry for soil sci-
ence is still far from being fully exploited. In order to utilize
the full potential of the NMR technique, it is necessary to
adapt it to the specific complexity and heterogeneity of soils
to gain a more detailed understanding of interaction dynam-
ics and soil-specific processes. Related NMR methods for
pore size and water distribution in soils evaluation are stray
field STRAFI NMR that uses a strong magnetic field gradi-
ent in a high field superconducting magnet, as well as pulsed
field gradient PFG NMR measurements for diffusion coeffi-
cients determination. The latter is especially promising in
combination with NMR relaxometry [60, 64, 95, 96] to relate
relaxation time with molecular diffusivity. During the last
ten years mobile NMR devices have been developed in order
to be able to investigate porosity and water distribution in
samples in-situ with devices that promise easy handling.
Several different approaches should be mentioned here, in-
cluding the NMR MOUSE (mobile universal surface ex-
plorer) a unilateral scanner [29, 30, 35-37] and in-side-out
NMR devices like the Hallbach Scanner for bore hole appli-
cations [2, 31-34]. Field cycling NMR techniques span a
wide range of magnetic fields and, therefore, proton Larmor
frequencies (10 kHz-40 MHz) within one single instrument.
These techniques are a powerful and promising tool to study
interaction dynamics [39]. Although the fields of application
do not yet span investigation of soil samples, especially field
cycling NMR will help to gain valuable complementary de-
tailed understanding on interactions between soil and water.
The development of new T
1
pulse sequences reducing the
overall measurement time may lead to a more frequent use of
T
1
measurements. This may further improve the understand-
ing of soil-water interactions as T
1
probes more directly in-
teractions of the spin system (i.e. water) and the environment
(i.e. the pore surface).
The potential of NMR relaxometry lies in the strong sen-
sitivity of relaxation times to numerous factors relevant in
soil-water-organics interactions, which is, however, at the
same time disadvantageous and hides the danger of severe
misinterpretations especially in systems as complex, versa-
tile and heterogeneous as soils. It, thus, has to be kept in
mind that conclusions on soil processes have to be drawn
with care and on the basis on detailed targeted process analy-
sis.
ABBREVIATIONS
DSC = differential scanning calorimetry
EPS = extra cellular polymeric substances
FA = fulvic acid
HA = humic acid
MOUSE = mobile universal surface explorer
MRI = magnetic resonance imaging
NMR = nuclear magnetic resonance
NOM = natural organic matter
PFG NMR = pulsed field gradient NMR
RF = radio frequency
SOM = soil organic matter
STRAFI NMR = stray field NMR
ACKNOWLEDGEMENTS
We like to thank Hella Korn, Dr. Uta Bckelmann and
Daniel Wicke from the TU Berlin for their help with the
biofilm reactors. This study was part of the projects
1 10 100 1000
0
4
8
12
Peak 3 Peak 2
Peak 1
A
m
p
l
i
t
u
d
e

[
a
.
u
.
]
T
2
[ms]
without biofilm
biofilm 1
biofilm 2 (clogged)
SCHA849/5 and SCHA849/8 funded by the German Re-
search foundation (DFG).
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Received: May 04, 2009 Revised: November 17, 2009 Accepted: January 25, 2010

Bayer et al.; Licensee Bentham Open.


1874-7698/10 2010 Bentham Open
Open Access
Proton NMR Relaxometry as a Useful Tool to Evaluate Swelling Processes
in Peat Soils
Fabian Jaeger
a
, Anastasia Shchegolikhina
b
, Henk Van As
c
and Gabriele Ellen Schaumann*
,a

a
Department of Environmental and Soil Chemistry, Institute of Environmental Sciences, Universitt Koblenz-Landau,
Fortstr. 7, 76829 Landau, Germany
b
Geographical Institute, Ruhr-University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany.
c
Laboratory of
Biophysics and Wageningen NMR Centre, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
Abstract: Dramatic physical and physico-chemical changes in soil properties may arise due to temperature and moisture
variations as well as swelling of soil organic matter (SOM) under constant conditions. Soil property variations may influ-
ence sorption/desorption and transport processes of environmental contaminants and nutrients in natural-organic-matter-
rich soils. Notwithstanding the studies reported in literature, a mechanistic model for SOM swelling is unavailable yet.
The objective of the present study was the evaluation of the swelling of peat soils, considered as SOM models, by
1
H
NMR relaxometry and differential scanning calorimetry (DSC). Namely, information on the processes governing physical
and physicochemical changes of peat during re-hydration were collected. The basic hypothesis of the present study was
that the changes are slow and may affect water state as well as amounts of different water types into the peats. For this
reason, such changes can be evidenced through the variations of mobility and thermal behaviour of the involved H
2
O
molecules by using
1
H NMR relaxometry and DSC. According to the experimental results, a mechanistic model, describ-
ing the fundamental processes of peat swelling, was obtained. Two different peats re-wetted at three temperatures were
used. The swelling process was monitored by measuring spin-spin relaxation time (T2) over a hydration time of several
months. Moreover, DSC, T1 T2 and T2 D correlation measurements were done at the beginning and at the end of the
hydration. Supplementary investigations were also done in order to discriminate between the swelling effects and the con-
tributions from soil solution, internal magnetic field gradients and/or soil microorganisms to proton relaxation. All the re-
sults revealed peat swelling. It was evidenced by pore size distribution changes, volumetric expansion and redistribution
of water, increasing amounts of nonfreezable and loosely bound water, as well as formation of gel phases and reduction of
the translational and rotational mobility of H
2
O molecules. All the findings implied that changes of the physical and phys-
icochemical properties of peats were obtained. In particular, three different processes having activation energies com-
prised in the interval 5 50 kJ mol
-1
were revealed. The mechanistic model which was, then, developed included water
reorientation in bound water phases, water diffusion into the peat matrix and reorientation of SOM chains as fundamental
processes governing SOM swelling. This study is of environmental significance in terms of re-naturation and re-watering
of commercially applied peatlands and of sorption/desorption and transport processes of pollutants and nutrients in natural
organic matter rich soils.
Keywords: Soil organic matter, swelling, kinetics, unfreezable water, differential scenning calorimetry, NMR relaxometry,
pore water.
INTRODUCTION
Natural soils are exposed to dynamic variations in tem-
perature and moisture. Changes in moisture status may affect
soil properties like water content or volumetric swelling of
soil organic matter (SOM) [1, 2] or sorbent properties [2-5].
Furthermore, the extractability of organic pollutants was
found to be affected by the hydration time of soils indicating
physical and physicochemical changes of SOM upon swel-
ling [2]. Although it is known that SOM swelling includes
volumetric expansion [1], water redistribution and re-
opening of small-sized pores [6] as well as SOM re-
organisation [7, 8] a mechanistic model describing the

*Address correspondence to this author at the Department of Environmental
and Soil Chemistry, Institute of Environmental Sciences, Universitt,
Koblenz-Landau, Fortstr. 7, 76829 Landau, Germany; Tel: +49(6341) 280-
571; Fax: +49(6341) 280-576; E-mail: schaumann@uni-landau.de
fundamental processes of SOM swelling is not available un-
til now.
Peat soils may be considered as concentrated analogues
of SOM, and, therefore, they were used as models for envi-
ronmental studies of pollutant sorption and transport in SOM
[9]. Peats are the product of accumulation and humification
of plant material in certain special wet habitats [7, 9], and are
commercially used as fuel and horticulture medium [7].
Beside peats, many macromolecular substances, such as
synthetic polymers or biopolymers, swell when placed in
contact with fluids. The swelling is characterised as sorption
of the liquid into the macromolecular solid phase, which in
turn, increases its volume. This volumetric swelling, Q, was
defined as the ratio of swollen to non-swollen volume of the
sorbent or macromolecular material; a value of unity indi-
cates no swelling. The amount of swelling depends both on
the nature of fluid and sorbent. Strongly cross-linked materi-
28 The Open Magnetic Resonance Journal, 2010, Volume 3 Jaeger et al.
als swell less than weakly cross-linked materials. However,
swelling seems to be limited by molecular size exclusion
effects to fairly small liquid molecules with molar volumes
smaller than about 93 cm
3
mol
1
for most soil organic mate-
rials and 88 cm
3
mol
1
for cellulose. The Q values for differ-
ent materials after water sorption ranged between 1.3 and 1.6
for peat, 2.0 for cellulose and 1.6 for chitin. [1]
Unlike synthetic polymers, McBrierty et al. [7, 8] re-
ported that the peat matrix re-organises during swelling,
thereby permitting access to a greater number of water mole-
cules to the hydrophilic sites. Furthermore, up to four differ-
ent types of water in hydrated peat samples were identified
using differential scanning calorimetry (DSC), thermogra-
vimetric analysis (TGA) and proton nuclear magnetic reso-
nance (NMR) relaxometry. They differentiated between
tightly bound or non-freezable water, up to two types of
loosely bound or freezable water and freezable bulk water.
Non-freezable water is predominantly hydration water and/or
water that interacts chemically with hydrophilic moieties in
the matrix showing glassy behaviour with a transition tem-
perature around -123C to -83C [7]. The character of
loosely bound water deviates less dramatically from that of
normal water. This water melts with further increase in tem-
perature, but at lower temperatures than freezable bulk water
that melts around 0C [7, 10]. In hydrogels, at least three
kinds of water were determined: hydrated water, interfacial
water, with a certain ordered arrangement, and bulk water
[11].
Additionally to the calculation of Q,
1
H NMR relaxome-
try [2, 6, 7, 12, 13] and DSC [7, 14] were used to study the
hydration of peat and mineral soil samples or to characterise
different types of water in peats. DSC was used to study the
melting and freezing behaviour of water and to calculate the
amounts of non-freezable and freezable water in hydrogels
[15, 16] or peat soil samples [7, 14]. The melting of
freezable water in moist samples is characterised by endo-
thermic water melting peaks in the DSC thermograms, which
originate from the additional energy uptake of the melting
process. The enthalpies of these peaks are often smaller than
it would be expected regarding the amount of water present
in the samples, which is due to the existence of non-
freezable water. In general, the melting peaks of water in
hydrogels and peat differ strongly from those of free pure
water or solutions. They are broader and split into two or
more overlaying peaks. Radosta and Schierbaum found a
splitting of the melting peak of water only in maltodextrin
gels, but not in maltodextrin solutions, although the deter-
mined amounts of non-freezable water were comparable for
both systems [15, 17]. Consequently, they attributed the
melting peak splitting to the entrapment of water within the
gel matrix. In peat soil samples, the splitting of the melting
peak was ascribed to the existence of loosely bound and
freezable bulk water [7, 14].
Swelling kinetics experiments of soil samples using
1
H
NMR relaxometry have shown that the swelling process is
linked to the migration of the peak relaxation times towards
smaller relaxation times and the increase of the amount of
water protons relaxing at smaller relaxation times [2, 6, 12,
13]. These findings were attributed to a redistribution of wa-
ter during swelling. Todoruk et al. [6] identified two proc-
esses with time constants of about 1 d for a fast process and
up to 22 d for a slow process for different soils, which were
comparable to the values reported by Schaumann et al. [2,
12] and Jaeger et al. [13]. The calculated activation energies
of these two processes ranged from 14 kJ mol
-1
to 117
kJ mol
-1
suggesting diffusion processes and chemical reac-
tions, such as ester hydrolysis [6]. The slow process was
attributed to the formation of gel phases and to the slow wa-
ter intrusion into micropores, which re-opened during swel-
ling.
The changes in the relaxation time distributions during
swelling of soils were found to be pronounced stronger in
soils with higher microbial respiratory activity [13] and in
soils inoculated with a biofilm producing bacteria isolate
[18]. Consequently, soil microorganisms may contribute to
the swelling of soils due to the production and release of
extracellular polymeric substances (EPS) and the formation
of biofilm. Furthermore, the proton relaxation in soil solu-
tions (bulk relaxation) may significantly contribute to the
total proton relaxation of water in soil samples and the con-
tribution of the bulk relaxation may increase during hydra-
tion due to dissolution of paramagnetic iron and manganese
or changes of their chemical speciation [19]. Keating and
Knight [20] found for iron-oxide coated sands that some of
the studied surface species of iron produced internal field
gradients causing additional transverse proton relaxation due
to spin diffusion in internal field gradients at 2.2 MHz. Thus,
a contribution of this additional transverse proton relaxation
mechanism cannot be excluded a priori for hydrated soil
samples measured at lower magnetic field strength.
In general, transverse relaxation times (T
2
) are used to
study the swelling of soil samples [6, 12, 13], because of the
much faster determination compared to longitudinal relaxa-
tion times (T
1
). However, many swellable materials, such as
cellulose [21], hydrogels with different degrees of cross-
linking [22], wheat starch [23] and maltodextrin [17] show
strong differences between T
1
and T
2
. Their T
1
/T
2
ratios were
found to be much larger than for free pure water (T
1
/T
2
~ 1)
or for rock core samples with T
1
/T
2
ratios generally between
2 to 3 [24]. The larger T
1
/T
2
ratios in those swellable materi-
als were attributed to a water structuring caused by the
polymer, which resulted in reduced rotational mobility of
water molecules inside the structured water phase [17]. This
reduced rotational mobility affects T
1
and T
2
differently [25].
The structured water is non-freezable water [26, 27] that
exchanges very fast with the bulk water phase, which results
in only one average relaxation time much smaller than for
bulk water [23]. In starch pastes, the T
1
/T
2
ratio of the bound
water was found to be 22 and the one determined from the
average relaxation times was about 8 [23]. As a conse-
quence, both T
1
and T
2
measurements may be helpful to in-
vestigate water mobility and a possible structuring of water
caused by the SOM matrix in swollen soils.
We hypothesised that the physical and physicochemical
properties of peat soil samples change during volumetric
swelling at constant temperature and moisture conditions.
These changes are slow and affect the state of water as well
as the amounts of different water types inside the peats.
Thus, they can be observed through changes in mobility and
thermal behaviour of the involved water molecules, which
Swelling of Peat Studied by 1H NMR Relaxometry The Open Magnetic Resonance Journal, 2010, Volume 3 29
can be determined by
1
H NMR relaxometry and DSC. Our
objectives were to study the swelling of peat soils via
1
H
NMR relaxometry and DSC to characterise the governing
processes causing physical and physicochemical changes of
peat during re-hydration at constant temperature and mois-
ture conditions. For that we have combined T
1
and T
2
meas-
urements together with DSC measurements for the first time
to study the swelling of peat soil samples. Swelling kinetics
experiments were carried out with two re-wetted peats at
three different temperatures. Additional investigations were
carried out to distinguish between swelling effects and fur-
ther influences on the proton relaxation process caused by
the soil solution, internal magnetic field gradients and/or soil
microorganisms.
MATERIALS AND METHODOLOGY
Peat Soil Samples
The peat material used in this study was taken from the
peat land Totes Moor in the nature park Steinhuder
Meer near Hannover, Germany. The samples of peat were
collected from the drained part of the bog from two different
layers: a fibric peat (peat 1) was collected from an upper
layer with low degree of decomposition and a well decom-
posed sapric peat (peat 2) was taken from 1.1 m depth. After
sampling, the peats were air dried at room temperature and
characterised (Table 1). For NMR measurements, the air
dried peat samples were ground, 2 mm sieved and stored at
19C until further usage.
Table 1. Description and Some Properties of the Two Studied
Peat Soil Samples. CEC
eff
Effective Cation Exchange
Capacity. DOC Dissolved Organic Carbon. wc
air dried

Gravimetric Water Content of the Air Dried Peat
Samples Related to Dry Mass (d.m.) Determined
after Oven Drying for 24 h at 105C
Fibric Peat Sapric Peat
Label Peat 1 Peat 2
Color Brown Black
Sampling depth /m 0.4 1.1
Ash-content /% 1.25 1.28
pH (CaCl2 0.01 M) 2.7 2.7
Corg /% 45 52
C/N 405 219
DOC /mg L
-1
89 56
CECeff /mmolc kg
-1
166 123
wcair dried /g g d.m. 0.152 0.161
Sampling coordinates 52 30' 26.41'' N
9 21' 14,28'' E

1
H NMR Relaxation in Porous Media
In porous materials, the proton relaxation is strongly ac-
celerated by interactions between water protons and surfaces
[detailed information can be found in e.g. 12, 28-30]. As a
result, the measured relaxation time of water protons is de-
termined by the proton relaxation time in the pore space,
T
1,2B
, the surface relaxation time, T
1,2S
, and the diffusion re-
laxation time, T
2D
, due to spin diffusion in internal magnetic
field gradients, which affects the transverse proton relaxation
time (T
2
), but not the longitudinal proton relaxation time (T
1
)
[28, 31].
1
T
1
=
1
T
1B
+
1
T
1S
=
1
T
1B
+

1
S
V
(1)
1
T
2
=
1
T
2B
+
1
T
2S
+
1
T
2D
=
1
T
2B
+

2
S
V
+
1
T
2D
(2)
With SV
1
= r
1
the proton relaxation time is connected
to the pore diameter, d
pore
= 2r , where r is the pore radius and
= 1, 2, or 3 is the shape factor for planar, cylindrical, and
spherical pore geometry, respectively [32]. SV
-1
is the pore
surface to pore volume ratio,
1, 2
is the surface relaxivity,
which is strongly affected by paramagnetic ions on the sur-
face like Mn(II) [33] and Fe(III) [34]. T
2D
is related to the
average internal gradient of the magnetic field, G, and the
self-diffusion coefficient of water, D, by
1
T
2D
=
D
12
G t
E
( )
2
,
(3)
where is the gyromagnetic ratio, and t
E
is the echo time,
which is the time between two 180 pulses in the Carr-
Purcell-Meiboom-Gill (CPMG) pulse sequence [35].
Eq. 1 and 2 are valid if the condition for the fast-diffusion
regime,
2
V
SD
<< 1
is fulfilled [31]. This means that the relaxa-
tion process is surface-limited and the diffusion of water
protons towards the surface is very fast and can therefore be
neglected. Various rock core samples, e.g. sandstones [28,
36], were assigned to the fast-diffusion regime [31], whereas
some sandy soil samples [37] were in the transition from the
intermediate- to the slow-diffusion regime [31]. However, it
has been shown that Eq. 2 can be also used for the calcula-
tion of pore sizes in soils by assuming the fast-diffusion re-
gime in these soil samples and by using two surface relaxiv-
ities for each soil, one for micro- and one for mesopores,
[37].
Sample Preparation and
1
H NMR Measurements
Sample Preparation
1.00 g dry mass (d.m.) of peat 1 and 3.55 g d.m. of peat 2
of the air dried samples were filled in glass vessels and
rewetted to their maximal water holding capacity (i.e. maxi-
mal water content). The maximal water content was 15.67 g
g
-1
d.m. for peat 1 and 4.51 g g
-1
d.m. for peat 2. Thus, the
total amount of water was with 15.67 g (peat 1) and 16.01 g
(peat 2) comparable for the two rewetted peats.
The protocol of the rewetting procedure was the same for
all studied peat samples: After placing the air dried peat
sample into the glass vessel, the water (demineralised water)
was added and the peat was carefully mixed with the water
using a thin spatula. After wetting of all surfaces, i.e. the
peat surfaces turned dark and no dry areas were observed,
the vessel was sealed with a plastic lid and knocked ten
times vertically on a solid surface to obtain comparable bulk
densities. After centrifugation at 4000 RPM at the beginning
and end of swelling (see below), the contact angle of the peat
samples was determined using the sessile drop method de-
scribed by Diehl and Schaumann [38] to test the wettability
of the surfaces. Before starting the rewetting procedure, both
the peat sample and the water were adjusted to the respective
temperature of 5C, 19C or 30C. In the course of swelling
of 5 to 7 months (hydration time), the sealed glass vessels
were stored in desiccators with 99.9 % relative humidity in
darkness at the respective temperature ( 1.5C).
Swelling Kinetics at 5C, 19C and 30C by
1
H NMR
Measurements
All one-dimensional
1
H NMR measurements to deter-
mine the swelling kinetics, the water distribution versus cen-
trifugation speed as well as the amounts of unfrozen water at
-34C and -5C were performed at a magnetic field strength
of 0.176 T, i.e. at a proton Larmor frequency of 7.5 MHz
(Minispec 7.5, Bruker, Germany). The temperatures during
the measurements were kept constant ( 0.5C) using a
XR401 Air-Jet
TM
sample cooler (FTS Systems, Stone Ridge,
USA) with compressed air as gas stream. The relaxation time
distributions were determined using a MATLAB program
developed by Veevaete [39] applying the BRD (Butler,
Reeds and Dawson) algorithm [40]. The relaxation time dis-
tributions consisted of 200 time constants (TC) with associ-
ated amplitudes. The sum of the amplitudes equals the total
NMR signal intensity at time 0 = t ( SI
t =0
), which is a meas-
ure of the total amount of water protons in a sample. All one-
dimensional
1
H NMR measurements were performed with
duplicates of the respective peat samples. In this study, we
present the results of the two repetition samples (RS 1 and
RS 2) separately, because the reproducibility between the
duplicates was partly not given.
The two-dimensional T
1
- T
2
and T
2
- D correlation meas-
urements were performed at a proton Larmor frequency of
30 MHz at 20 1C using a home-made NMR spectrometer
controlled by a MARAN Ultra console (Resonance In-
struments Ltd, Oxfordshire, UK) with an additional pulsed
field gradient (PFG) unit. For T
1
- T
2
correlated measure-
ments the inversion recovery method was combined with a
CPMG sequence. 25 steps of the inversion recovery time
between 750 s and 15 s were used to sample a T
1
relaxation
decay curve. In the T
2
- D correlation measurement a diffu-
sion-weighted multi-spin-echo pulse sequence [41] was used
to determine the correlation between T
2
and the self-
diffusion coefficient of water in the peat samples. 25 gradi-
ent steps with a maximal gradient strength of 1.2 T m
-1
were
used. Small and big delta were 1 ms and 10 ms, respectively.
Two-dimensional T
1
- T
2
and T
2
- D correlation was ana-
lyzed by 2D ILT two-dimensional numerical inverse
Laplace transformation [42-44].
The longitudinal relaxation time (T
1
) distributions of wa-
ter in the peat soil samples were determined using an inver-
sion recovery (IR) sequence with 25 IR-points between 1.0
ms and 19 s. Because of the long measurement time for T
1
of
about 75 min, the T
1
distribution was only determined direct
after water addition (after 110 - 180 min) and at the end of
the swelling experiment after 5 to 7 months. A CPMG pulse
sequence [35] with an echo time of 500 s and 50,000 ech-
oes was used to obtain transverse relaxation time (T
2
) distri-
butions of water in the peat soil samples in the course of peat
swelling. A total of 30 to 36 T
2
measurements were per-
formed to follow the peat swelling over 5 to 7 months. Dur-
ing the first 30 minutes after water addition, T
2
measure-
ments were performed every five minutes. Within the first
three to seven hours after water addition, a total of up to
eight T
2
measurements were performed to determine fast
changes in the T
2
distributions. The effect of the additional
transverse relaxation due to spin diffusion in internal field
gradients was tested according to Keating and Knight [20]
determining T
2
as a function of increasing echo times (500 to
800 s).
Cryo - NMR Relaxometry: Determination of
Non-Freezable and Loosely Bound Water
1
H NMR relaxometry experiments at -34C and -5C
were carried out to determine the amounts of water that were
left unfrozen in the peats at these temperatures. The first
represents the lowest temperature that was reachable in the
peats using the Air-Jet
TM
sample cooler. The unfrozen water
at -34C is referred to as tightly bound or non-freezable wa-
ter [7] in this study. It was tested by DSC whether this water
remained unfrozen between -90C and -34C. From DSC it
was found that melting of soil solution extracted from the
hydrated peats started above -4C. Thus, the second tempera-
ture of -5C was operationally chosen to measure the
amounts of loosely bound water in peat [7]. This kind of
NMR measurement at low temperatures is referred to as
Cryo - NMR relaxometry hereafter. The amounts of these
two types of water in peat were determined from the NMR
signal intensity using a T
2
Hahn-echo [45] sequence with 25
echo points with increasing echo times between 0.05 ms and
7.5 ms at the beginning and end of peat swelling. T
2
of non-
freezable and loosely bound water was calculated by mono-
exponential fitting to the decay curves determined at -34C
and -5C, respectively. The wet peat samples were first
cooled down to -34C inside the NMR probe using the Air-
Jet
TM
sample cooler. The freezing process of water was char-
acterised by a dramatic decrease of the NMR signal inten-
sity, because the very short T
2
of ice of about 10 s [46]
could not be detected due to the dead time of 50 s of the
used NMR device. The signal intensity was constant after
about 60 min of cooling and the Cryo-NMR measurement
was carried out. After that the sample was adjusted to -5C
until constant signal intensity was observed and subse-
quently measured. Two additional samples (prepared as de-
scribed above) were used to determine the non-freezable and
loosely bound water at the beginning of peat swelling. For
the determination of both water types at the end of swelling,
the original samples from the swelling kinetics at 30C were
used.
Water Distribution Versus Centrifugation Speed
The water distribution in the peat samples versus cen-
trifugation speed was determined at the beginning and end of
swelling and after 2 days of hydration (no repetition sample)
using a Universal 320 centrifuge with the rotor 1494 (Het-
tich, Tuttlingen, Germany). The original samples from the
swelling kinetics at 30C were used to determine the water
distribution at the end of swelling. Additional repetition
samples were used for the determination of the water distri-
bution at the beginning of swelling and after 2 days of hydra-
tion. The peat samples were re-wetted as described above
and filled into centrifuge tube filters with a 10-m filter in-
sert (VectaSpin 20, Whatman). Then they were centrifuged
step-wise at nine centrifugation speeds between 500 RPM
and 4000 RPM for 15 min at 19C. The peat samples were
desaturated during centrifugation. After every centrifugation
step, the gravimetric water contents and the T
2
and T
1
distri-
butions of water in the peat samples at 19C were deter-
mined. Instead of the largest T
2
and T
1
in the relaxation time
distribution, T
2
and T
1
at 90 % of total sum of amplitudes
were determined to reduce variation between replicates [37].
After the last centrifugation step, T
1B
and T
2B
were measured
in the extracted soil solution, which was gained during cen-
trifugation, to determine the contribution of the soil solution
to the total proton relaxation in the wet peat samples.
T
1
/T
2
Ratios as a Measure of the Water State in Peat
Samples
T
1
/T
2
ratios were determined to characterise the state of
water in peat samples. For that, we calculated the T
1
/T
2
ra-
tios from the relaxation time distributions of water in peat of
the fully water saturated samples and the step-wise desatu-
rated samples after centrifugation at the beginning and end
of hydration time as a function of the relative water content
(rel. wc). Relative water contents in the fully saturated sam-
ples were calculated from the quotient of the subtotal of the
amplitudes (AT) of the 200 time constants (TC) in a relaxa-
tion time distribution and the total sum of the amplitudes
( SI
t =0
)
rel.wc =
AT
i
i =1
200
SI
t =0
100.
(4)
The T
1
/T
2
ratios in the saturated samples were deter-
mined from TC in the T
1
and T
2
distributions at 25 relative
water contents between 7.5 % and 100 %. In the desaturated
samples, relative water contents were calculated from the
quotient of the gravimetric water content after step-wise cen-
trifugation (wc
cen.
) and the total water content at saturation
(wc
tot
)
rel.wc =
wc
cent
wc
tot
100.
(5)
T
1
/T
2
ratios in these samples were determined from T
2

and T
1
at 90 % of total sum of amplitudes after every cen-
trifugation speed (see above).
DSC Measurements
The freezing/melting behaviour of water inside the wet
peat samples was studied between -90C and 40C using a
TA Instruments Model Q1000 DSC (TA Instruments, Al-
zenau, Germany) with nitrogen as purge gas (50 mL min
-1
).
Heat flow and temperature calibration were carried out using
Indium. For the DSC measurements ~3 mg - 10 mg of wet
and partly desaturated peat soil from the samples used in the
centrifugation experiment were weighed into aluminium
pans and hermetically sealed using an aluminium lid. The
cooling/heating protocol started with an equilibration at
40C followed by a cooling cycle down to -90C with cool-
ing rates of 3 K min
-1
until -75C followed by 2 K min
-1
until
-90C. After equilibration at -90C, the sample was heated
up to 40C with a heating rate of 5 K min
-1
. Data analysis
(determination of peak onset and peak maximum tempera-
tures, and melting enthalpy) was performed using the Uni-
versal Analysis 2000 software by TA Instruments. The peak
maximum temperature is referred to as peak position is this
study.
According to [14], the melting peak was investigated at
four different heating rates between 0.5 K min
-1
and 10
K min
-1
to test whether the melting transition is kinetically
controlled. For that, the desaturated peat samples from the
centrifugation experiment at 4000 RPM, agar gel (30 g L
-1
),
water saturated sand with particle sizes of 150 200 m
(referred to as sand 150 m) and free pure water were used.
The melting enthalpy of freezable water,
H
f
, in the
peat samples as well as the amount of non-freezable water
were calculated from the DSC thermograms of the desatu-
rated peat samples from the centrifugation experiment. Ac-
cording to McBrierty et al. [7], both were calculated from
the integrated change in enthalpy (per gram of dry peat)
which was plotted as a function of water content. The slope
of a fitted straight line provides
H
f
and the intercept on
the water content axis is the amount of non-freezable water.
Microbial Respiratory Activity
For the incubation experiment the samples were placed in
vessels and wetted to 60 % water holding capacity. The
rewetted peats (as triplicates) were incubated for 21 days at a
temperature of 20C in a Respicond-apparatus (Nordgren
Innovations, Bygdea, Sweden), which determined the CO
2
-
evolution hourly from the changes in electrical conductivity
in 10 ml of 0.6 M KOH solution placed inside the incubation
vessels [47].
Calculation of Time Constants and of Apparent
Activation Energy the Rate-Limiting Step of Peat Swelling
The T
2
distributions of water in the peat samples used in
the swelling kinetics experiment were operationally divided
into four T
2
ranges with fixed T
2
limits (I: 0.1 - 3 ms; II: 3 -
30 ms; III: 30 - 300 ms and IV: > 300 ms). The relative
amplitudes of the four T
2
ranges were calculated (i.e. the
sum of amplitudes of the respective T
2
range divided by the
total sum of amplitudes or SI
t =0
) and plotted as a function of
hydration time. Previous studies have shown that swelling
kinetics of soil samples can be described by first-order proc-
esses using exponential functions [12, 13]. We tested fittings
with up to four exponential functions to describe the time
dependent change of the relative amplitudes A(t ) of the T
2

range IV (T
2
> 300ms), which represented the intrusion of
water into the peat matrix, and to calculate the time constants
of the rate-limiting processes of peat swelling. The best re-
sults (large R
2
, small standard errors and good reproducibil-
ity between replicates) were determined using a sum of three
exponentials
A(t ) = A
i
exp
t
t
i
_
,
i =3
+ A
~
.
(6)
A
i
(i = 3) is the relative amplitude of each exponential
function,
A the relative amplitude for infinite hydration

time, t the hydration time [d],
i
(i = 3) the time constant of
each first order process [d]. The reciprocal,
1
i
(i = 3)
, repre-
sents its rate constant, k, [d
-1
].
According to the Arrhenius-Equation, the temperature
dependency of the rate constant, k, of the water intrusion
allows to calculate the activation energy, E
A
, which is the
minimum energy necessary for a specific process to occur
[48]
ln(k) = ln(A
*
)
E
A
R
1
T
. (7)
A
*
is the pre-exponential factor (commonly written as
A, but changed to A
*
to avoid confusion with A used for
relative amplitude), R the universal gas constant
( = R 8.314472 J mol
1
K
1
), T the absolute temperature (K).
The apparent activation energy gives information about the
nature of the rate-limiting step of the investigated process.
Chemical reactions are characterised by E
A
> 60 kJ mol
-1
,
while physically controlled processes require E
A
< 40 kJ mol
-1

[49].
RESULTS AND DISCUSSION
Changes of Relaxation Time Distributions During
Hydration
The T
1
and T
2
distributions of water protons in the two
rewetted peat samples at the beginning and end of the swel-
ling experiment are shown in Fig. (1) for peat 1 and Figure 2
for peat 2 for the temperatures 5C (B+D) and 30C (A+C).
All relaxation time distributions consist of several peaks (up
to five peaks for peat 2), which are in some cases hardly dis-
tinguishable. However, the relaxation time distributions in
Figs. (1 and 2) show three features. Firstly, the shapes of the
T
1
and T
2
distributions at the end of the hydration period are
different to those directly after water addition (referred to as
start in the Figures). The relative amplitudes at smaller T
1

and T
2
values increased and those at larger T
1
and T
2
values
decreased for both peat samples. Parallel to this, the T
2
val-
ues of the peak maxima increased for peaks at smaller T
2

Fig. (1). T
1
and T
2
distributions of water protons in the rewetted peat 1 samples at the beginning and end of the swelling experiment deter-
mined at 5C (B+D) and 30C (A+C). The results of the two repetition samples or replicates (RS1 and RS2) are displayed.
5C
0.1 1 10 100 1000
0
1
2
3
4

peat 1 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T1 /ms
B
0.1 1 10 100 1000
0
1
2
3
4

peat 1 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T2 /ms
C
0.1 1 10 100 1000
0
1
2
3
4

peat 1 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T2 /ms
D
0.1 1 10 100 1000
0
1
2
3
4

peat 1 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T1 /ms
A
30C

(< 20 - 200 ms) and decreased for peaks at larger T
2
(> 200
ms). Secondly, the shapes of the T
1
distributions differ from
the shapes of the T
2
distributions. Thirdly, the width of the
T
1
and T
2
distributions for peat 2 are found to be broader
than those of peat 1. Furthermore, the relative amplitudes at
T
1
< 100 ms and at T
2
< 10 ms are larger for peat 2.
The sample weights and the total sum of amplitudes
( SI
t =0
) during hydration time of 5 to 7 months was constant
within 2 % for all peat samples. After calibration with pure
water at the different temperatures, SI
t =0
was used to calcu-
late the NMR detectable amounts of water in the peat sam-
ples, which were comparable to those determined by gra-
vimetric measurements. Hence, all water inside the peat
samples was determined by the
1
H NMR relaxometry meas-
urements and no water was lost during the long hydration
times. In the course of hydration, the peat sample volumes
increased and the calculated Q values were 1.5 for peat 1 and
2.0 for peat 2.
The relaxation time distributions of water in the two
repetition samples of each peat (referred to as RS 1 and RS 2
in Figs. (1 and 2) were similar with each other for all hydra-
tion time points. The only exception was the T
1
distribution
for the second repetition sample of peat 2 at 30C (RS 2 in
Fig. 2A), which was not comparable to the one for RS 1
(Fig. 2C).
Figs. (1 and 2) also show that the relaxation time distri-
butions were affected by the incubation temperature. An
increase of the peak relaxation times and of peak widths at
higher temperatures was found. Eq. 7 was used to calculate
the activation energies, E
A
, of the peak relaxation time in-
crease at the beginning and end of hydration time using
T
2
1
= k . E
A
was 4 - 12 kJ mol
-1
for peat 1 and 3 - 16 kJ mol
-1

for peat 2. T
2
of pure water increased from 1.5 s at 5C to 2.8
s at 30C and E
A
of this T
2
increase was 16.3 0.4 kJ mol
-1
.
Thus, the activation energies for the peat samples were
smaller then or comparable to E
A
of pure water. This sug-
gests that the temperature dependency of the relaxation time
distribution of water in the peat samples can be attributed
mainly to increasing water diffusivity. Furthermore, the posi-
tive relation between peak relaxation time and temperature
suggests that the proton relaxation in the peat samples was
surface limited [50].
The large Q values indicated volumetric swelling [1] of
both peats, but to a larger extent for peat 2. The decrease of
the T
2
values for peaks at larger T
2
(> 200 ms) is consistent
with the findings reported elsewhere and suggests swelling
of peat [6, 12, 13]. The increase of the T
2
values of the peak
maxima for peaks at smaller T
2
(< 20 - 200 ms) is contrary to
Jaeger et al. [13] and Todoruk et al. [6]. However, a qualita-
tively comparable peak maxima shifting was also observed
during the swelling of starchy sago beads (data not shown).
The differences between the two repetitions of peat 2 at 30C

Fig. (2). T
1
and T
2
distributions of water protons in the rewetted peat 2 samples at the beginning and end of the swelling experiment deter-
mined at 5C (B+D) and 30C (A+C). The results of the two repetition samples or replicates (RS1 and RS2) are displayed.
0.1 1 10 100 1000
0
1
2
3
4
peat 2 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T2 /ms
D
0.1 1 10 100 1000
0
1
2
3
4

peat 2 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T1 /ms
B
0.1 1 10 100 1000
0
1
2
3
4

peat 2 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T2 /ms
C
0.1 1 10 100 1000
0
1
2
3
4

peat 2 RS1 RS2
start
end
r
e
l
.

a
m
p
l
i
t
u
d
e

/
%
T1 /ms
A
5C 30C

may be due to the two weeks longer hydration time of RS 2
and resulting stronger peat swelling, which may have af-
fected T
1
differently in the second repetition than in the first.
Water Distribution Versus Centrifugation Speed
The distribution of water in the two peat samples at the
beginning, after 2 days and at the end of hydration time are
represented as a function of centrifugation speed in Fig. (3).
Direct after water addition (start), centrifugation at 500 RPM
resulted in a dramatic decrease of the relative water contents
of the peats to about 40 % of the original water contents. No
significant changes of the relative water contents were ob-
served for the next one to two centrifugation speeds (peat 1
and peat 2, respectively). For centrifugation speeds faster
than 800 RPM (peat 1) or 1000 RPM (peat 2), the water con-
tents decreased with increasing centrifugation speed, with an
end point of ~15 % and ~25 % at 4000 RPM (peat 1 and peat
2, respectively). With increasing hydration time of the peat
samples, the relative water contents at all centrifugation
speeds increased. This observation was more pronounced for
smaller centrifugation speeds, where the relative water con-
tents at the end of hydration were almost two times larger
than at the beginning. The water contents after two days of
hydration for smaller centrifugation speeds were found to be
between the values determined at the beginning and the end
of hydration. For larger centrifugation speeds, they were
comparable to the initial values. The observed changes were
more pronounced for peat 2 than for peat 1.
The water distribution of RS 2 of peat 2 at the end of hy-
dration (Fig. 3B) was comparable to the one of RS 1 only for
centrifugation speeds faster 2000 RPM. For centrifugation
speeds slower 2000 RPM, the values of the relative water
content were significantly larger. For the other peat samples,
the two repetition samples were similar (day 2 was studied
without any repetition sample).
With the assumption that every centrifugation speed rep-
resents a certain pore size and the represented pore size de-
creases with increasing centrifugation speed [51], it can be
concluded that the pore size distribution changed signifi-
cantly during peat hydration. This change was slow and
takes place in two phases. The first phase started very soon
after water addition and lasted for more than two days. It was
characterised by the decrease of the number of very large
pores (> 50 m) represented by centrifugation speeds of
500RPM and the evolution of medium-sized pores 50 - 10
m [51], represented by centrifugation speeds of up to 1000
RPM. The second phase started anytime after the first two
days of hydration and resulted in the ongoing evolution of
medium-sized pores 50 - 10 m as well as of small-sized
pores 10 - 1 m [51], represented by centrifugation speeds of
1000 - 4000 RPM.
Fig. (4) shows the T
1
and T
2
relaxation times of water in
the desaturated peat samples as a function of centrifugation
speed (the same samples as presented in Fig. (3) without the
sample of day 2) to test whether the observed changes of the
relaxation time distributions in Figs. (1 and 2) were caused
by surface relaxivity changes of the peat samples during hy-
dration. The T
1
and T
2
relaxation times decreased with in-
creasing centrifugation speeds, but were comparable for the
beginning and for the end of hydration.
Again, the only exception was the sample 2 of peat 2
(Fig. 4B+D) for which T
1
values were always larger than
those of RS 1. In contrast, T
2
of RS 2 was larger only for
centrifugation speeds slower than 2500 RPM, but compara-
ble to those of RS 1 for larger centrifugation speeds. This
implies that both the transverse and the longitudinal surface
relaxivity decreased in RS 2 of peat 2. However, Fig.
(2A+C) show that only T
1
but not T
2
distributions are differ-
ent for the two repetition samples of peat 2 at 30C. It, there-
fore, is more likely that the two repetitions were different in
some physical properties which affected the dewatering by
centrifugation. For instance, reduced water conductivity due
to stronger water binding of entrapped water in a gel phase
may result in a less effective dewatering by centrifugation,
because smaller speeds cannot overcome the stronger water
holding. It was found that at high centrifugation speeds faster
2000 RPM only the T
1
but not the T
2
and the relative water
content values of the two repetition samples of peat 2 were
different. Hence, it can be suggested that only the longitudi-
nal, but not the transverse surface relaxivity of RS 2 of peat
2 at 30C increased during hydration. This may be due to a
stronger decrease of the water mobility in the vicinity of the
peat surface.

Fig. (3). Distributions of water in the two peat samples at the beginning, after 2 days and at the end of hydration time (the 30C samples at
the end of hydration measured at 19C) as a function of centrifugation speed. The results of the two repetition samples or replicates (RS1 and
RS2) are displayed (not for day 2).
1000
20
30
40
50
60
70
80
90 peat 1 RS 1 RS 2
start
end
day 2
4000 2000
A

r
e
l
.

w
c

/
%
centrifugation speed /RPM
500 1000
20
30
40
50
60
70
80
90 peat 2 RS 1 RS 2
start
end
day 2
4000 2000
B
r
e
l
.

w
c

/
%
500

For peat 1, the T
1
and T
2
values at 500 RPM were smaller
at the beginning than at the end of hydration (Fig. 4A+C).
The relative water content at the beginning of hydration was
constant between 500 RPM and 650 RPM (Fig. 3). Thus, the
smaller T
1
and T
2
values were due to only pores smaller than
represented by centrifugation speeds of 500 - 650 RPM were
existent in the desaturated samples direct after water addi-
tion. With the exception of RS 2 of peat 2 at 30C, the longi-
tudinal and the transverse surface relaxivities of the two peat
samples did not change significantly during hydration time.
Surface relaxivity changes may be only to some extent re-
sponsible for the observed changes of the T
1
distributions,
but not for those of the T
2
distributions in the course of hy-
dration of the two peats soil samples.
Calculation of Time Constants and of Apparent Activation
Energy
Fig. (5) shows the changes of relative amplitudes of the
four T
2
ranges (I: 0.1 - 3 ms; II: 3 - 30 ms; III: 30 - 300 ms
and IV: > 300 ms) in the T
2
distribution of water in the two
rewetted peat soil samples at 30C (Fig. 5A+C) and 5C
(Fig. 5B+D) as a function of hydration time. Within the first
hours after water addition, the relative amplitudes for T
2
>
300 ms decreased very fast followed by an ongoing slower
decrease, which lasted for several months. This slow de-
crease was not fully completed until the end of the available
experiment time of 5 to 7 months. Parallel to this decrease
the relative amplitudes of the T
2
ranges II and III increased
during the course of hydration. For peat 1 mainly the ampli-
tudes of the T
2
range III (30 - 300 ms) increased. For peat 2
the amplitudes of the T
2
range II (3 - 30 ms) increased. Simi-
lar to the decrease of the relative amplitudes for T
2
> 300 ms,
the increases of amplitudes at smaller T
2
were very fast in
the beginning and much slower after a few days lasting until
the end of the experiment.
From the contact angle experiment it can be concluded
that the peat surfaces were fully wettable after water addi-
tion, because the water drop needed for the contact angle
measurement did not rest at the peat surface, but was sucked
into the matrix immediately. The T
1B
and T
2B
bulk relaxation
times in the extracted peat soil solutions measured at 19C
were with 2.0 0.3 s comparable to that of pure water T
1,2B

= 2.3 s and did not change significantly during the course of
hydration for any of the two peats. Thus, the time dependent
changes of the relative amplitudes of the T
2
ranges repre-
sented mainly the intrusion of water into the peat matrix or
water re-distribution, but not surface wettability changes or
changes of the bulk relaxation times.
For both peats, three time constants in the range of min-
utes (fast), hours (medium fast) and several weeks to months
(slow) for the rate-limiting processes of water intrusion (de-
cease of the relative amplitudes for T
2
> 300 ms) were calcu-
lated with Eq. 6 (Table 2). The time constants of the three
processes were slightly larger for peat 1 and the relative
amounts of re-distributed water smaller than for peat 2. In

Fig. (4). T
1
and T
2
relaxation times of water in the two desaturated peat samples as a function of centrifugation speed (the same samples as
presented in Figure 3 without sample of day 2). The results of the two repetition samples or replicates (RS1 and RS2) are displayed.
1000
10
100
1000
peat 2 RS 1 RS 2
start
end
4000 2000
D
T
2

/
m
s
500 1000
10
100
1000
peat 1 RS 1 RS 2
start
end
4000 2000
C

T
2

/
m
s
500
1000
10
100
1000
peat 1 RS 1 RS 2
start
end
4000 2000
A

T
1

/
m
s
500 1000
10
100
1000
peat 2 RS 1 RS 2
start
end
4000 2000
B
T
1

/
m
s
500

total, about 20 % (peat 1) and 30 - 40 % (peat 2) of the total
water in the peat samples took part in the water intrusion. All
time constants decreased with increasing temperature, which
allowed the calculation of apparent activation energies, E
A
,
of the three rate-limiting processes of water intrusion using
Eq. 7. The activation energies (Table 2) were comparable for
the two peat samples and ranged between 5 kJ mol
-1
(fast
process), 15 - 25 kJ mol
-1
(medium fast process) and 40 - 50
kJ mol
-1
(slow process). Thus, E
A
of the medium fast process
is comparable to the value determine for pure water (see
above), whereas it is smaller for the fast, but larger for the
slow process.
The amounts of re-distributed water during hydration of
the peats were comparable to those found in mineral soil
samples [13]. The water re-distribution was accompanied by
a reduction of T
2
for 20 - 40 % of the total water, indicating a
decrease in mobility of the involved water molecules during
hydration [52]. The fast process of water re-distribution was
not observed in soil samples until now, which may be due to
the faster water addition of two instead of 15 minutes com-
pared to similar studies [12], the faster measurement of T
2

compared to T
1
[2] and the better time resolution within the
first minutes after water addition with one T
2
measurement
every five minutes compared to one measurement per day [2,
12, 13] or every 30 minutes [6]. The medium fast process
was also observed in other studies [6, 12, 13]. The time con-
stant of the slow process is up to 10 to 15 times larger than
reported elsewhere [2, 6, 12, 13]. The apparent activation
energies of the three processes are in the lower range as re-
ported by Todoruk et al. [6] or even smaller. As they [6]
found a negative relation between SOM content and the
value of the apparent activation energy, this may be due to
the high organic matter contents of about 99 % of the two
peats in this study. However, it cannot be excluded that dis-
solution of paramagnetic substances may have additionally
increased the values of the apparent activation energy in their
study, because the contribution of the soil solution to the
proton relaxation was not considered. It was shown in a pre-
vious study [19] that the concentration of iron and manga-
nese in the solution of peat is generally smaller than in the
solution of mineral soil samples and that the iron and man-
ganese concentration in soil solution may increase during
hydration.
The apparent activation energies calculated for the fast
process are comparable to the energy of 6.3 kJ mol
-1
required
to just break the hydrogen bond in a locally symmetric,
strongly H-bonded domain in water [53] leaving the mole-
cules essentially in the same position. E
A
of the medium fast
process is comparable to the value reported for water self-
diffusion [25], and indicates diffusion processes of water.

Fig. (5). Changes of the relative amplitudes of the four T
2
ranges (I: 0.1 - 3 ms; II: 3 - 30 ms; III: 30 - 300 ms and IV: > 300 ms) in the T
2

distribution of water in the two rewetted peat soil samples at 30C (Fig. 5A+C) and 5C (Fig. 5B+D) as a function of hydration time. The
results of the two repetition samples or replicates (RS1 and RS2) are displayed.
0 30 60 90 120 150 180
0
20
40
60
A

RS 1 RS 2 peat 1
0.1-3 ms
3-30 ms
30-300 ms
>300 ms
r
e
l
.

a
m
p
l
i
t
u
d
e

(
T
2
)

/
%
time /days
0 30 60 90 120 150 180
0
20
40
60
C

RS 1 RS 2 peat 2
0.1-3 ms
3-30 ms
30-300 ms
>300 ms
r
e
l
.

a
m
p
l
i
t
u
d
e

(
T
2
)

/
%
time /days
0 30 60 90 120 150 180
0
20
40
60
D

RS 1 RS 2 peat 2
0.1-3 ms
3-30 ms
30-300 ms
>300 ms
r
e
l
.

a
m
p
l
i
t
u
d
e

(
T
2
)

/
%
time /days
0 30 60 90 120 150 180
0
20
40
60
B

RS 1 RS 2 peat 1
0.1-3 ms
3-30 ms
30-300 ms
>300 ms
r
e
l
.

a
m
p
l
i
t
u
d
e

(
T
2
)

/
%
time /days
30C 5C
The larger activation energies of the slow process suggest
physical or physicochemical controlled processes, such as
water diffusion or reorientation of SOM chains during hydra-
tion [6, 38, 49].
T
1
- T
2
and T
2
- D Two-Dimensional NMR Measurements
The results of the T
1
- T
2
and T
2
- D two-dimensional
NMR measurements of the two peats at the beginning and
end of hydration time at 30 MHz and 20C are shown in Fig.
(6). Direct after water addition, most of the water protons
relaxed with T
1
and T
2
relaxation times comparable to pure
water, which is represented by the high intensity areas close
to the 1:1 line (white line) in Fig. (6A) for peat 1 and Fig.
(6D) for peat 2. However, some water protons at shorter re-
laxation times relaxed at smaller T
2
than T
1
values. This is
represented by the deviation of the intensity areas from the
1:1 line. This deviation was more pronounced at the end of
the hydration time (Fig. 6B+E), where the amount of water
protons relaxing at smaller relaxation times was increased
(Figs. 1, 2 and 5). Whereas the deviation from the 1:1 line
appeared to be continuously for peat 1 (Fig. 6B), it was more
step-wise for peat 2 (Fig. 6E). As a result of this deviation,
the T
2
values were about ten times smaller than the T
1
values
at small relaxation times. Furthermore, both figures show
that each T
1
is related to one T
2
value and vice versa. Thus,
each T
1
population was related to one T
2
population in the T
1

and T
2
distributions in Figs. (1 and 2), which allows a quanti-
tative comparison of the T
1
/T
2
ratios of water in the peat
samples used in the swelling kinetics experiment (see next
section).
The T
2
- D correlation spectra of water in the two peat
samples (RS 1 only) at the end of hydration at 20C are
shown in Fig. (6C) (peat 1) and Fig. (6F) (peat 2). For larger
relaxation times (T
2
> 30 ms), the apparent diffusion coeffi-
cient of water in the two peat samples was comparable to the
one of free water with D
app
= 2.03 x 10
-9
m
2
s
-1
[54]. For
smaller relaxation times (T
2
< 30 ms), the apparent diffusion
coefficient of water was reduced to D
app
= 1.2 x 10
-9
m
2
s
-1

for peat 1 and D
app
= 0.7 x 10
-9
m
2
s
-1
for peat 2. Thus, the
translational mobility of 22 % (peat 1) and 35 % (peat 2) of
the total water was reduced compared to that of free water.
At the beginning of hydration (data not shown), it was found
that the translational mobility of only 16 % (peat 1) and 12
% (peat 2) of the total water was reduced. This indicates that
the translational mobility of 6 % and 23 % of the total water
in peat 1 and peat 2 was reduced during hydration, respec-
tively. The reduced translational water mobility in the peats
may be due to the larger surface to volume ratios of smaller
pores [55] or due to water entrapment inside a gel phase,
where the water mobility is affected by the polymer chains,
as shown for e.g. agar gels [56].
The T
1
- T
2
correlation spectra of the two peats were very
different from those of sedimentary rock samples [42]. The
main difference is the stronger deviation of the high intensity
area from the 1:1 line for the peats. To compare this phe-
nomenon found in peat with other materials that are able to
swell and form gel phases, we studied additionally different
reference materials like agar, starch (1 - 2 mm sago beads)
and allophane gel (1.25 SiO
2
* Al
2
O
3
* 3.2 H
2
O). For agar
gel (c = 30 g L
-1
), the T
1
- T
2
correlation spectrum showed
one high intensity area in the shape of a circle at T
1
= 2000
ms and T
2
= 41 ms (data not shown). The resulting T
1
/T
2

ratio was about 50 at 30 MHz. Fig. (7A) shows that the T
1
/T
2

ratios of water in agar gels at 7.5 MHz strongly increase as a
function of the agar concentration. The T
1
/T
2
ratio in the agar
gel with c = 30 g L
-1
was with 27 smaller than at 30 MHz,
because T
1
was with 1240 ms much smaller than at 30 MHz
(T
1
= 2000 ms). T
2
was with 45 ms at 7.5 MHz comparable
to the value at higher field strength (T
2
= 41 ms). For inor-
ganic allophane gel (wc = 5.5 0.5 g g
-1
), the T
1
- T
2
correla-
tion spectrum was qualitatively comparable to the one of the
agar gel, but with smaller relaxation time values of T
1
= 330
ms and T
2
= 10 ms (data not shown). The calculated T
1
/T
2

ratio was 33 at 30 MHz and 25 at 7.5 MHz, because T
1
was
smaller at 7.5 MHz and T
2
similar to that at higher field
strength.
Fig. (7) also represents the T
1
- T
2
(Fig. 7B) and the T
2
-
D (Fig. 7C) correlation spectrum of water in swollen sago
beads. Sago is a starch extracted from the pith of sago palm
stems (Metroxylon sagu) and is commonly used in the food
industry. In contrast to agar and allophane gel, the T
1
- T
2

spectrum of swollen sago beads consist of a broad distribu-
tion, which is more similar to the one determined for peat 1
at the end of hydration. However, the deviation of the high
intensity area from the 1:1 line is stronger and the calculated
maximal T
1
/T
2
ratios are with 30 - 40 comparable to those of
the agar and allophane gels. The T
2
- D spectrum shows that
for some water at smaller T
2
the water mobility was with
D
app
= (0.5 - 1.8) x 10
-9
m
2
s
-1
smaller than for free pure wa-
Table 2. Results of the Peat Swelling Kinetics (i.e. Decrease of the Relative Amplitude of T2 > 300 ms) and Apparent Activation
Energies (E
A
) of the Three Determined Processes Governing the Swelling of peat. SE Standard Error
Sample Process Time Constant
(30C to 5C)
rel. Amount of Total
Water/%
EA SE/kJ mol
-1
fast 25-35 min 5-7 6 3
medium fast 20-50 h ~5 24 4
peat 1
slow 55-330 d 7-9 49 7
fast 15-20 min 10-17 4 2
medium fast 20-30 h 7-9 16 6
peat 2
slow 45-210 d 11-16 42 5
ter, which is comparable to peat 2 at the end of hydration.
Thus, the two-dimensional correlation spectra of the two
peats were qualitatively comparable to those of the swollen
sago beads suggesting similar properties in terms of proton
relaxation and water mobility.
For all peat samples and reference materials, T
2
was de-
termined as a function of echo time to estimate the contribu-
tion of T
2D
to the total proton relaxation. The T
2
distribution
did not change significantly with increasing echo times for
all samples. Furthermore, T
2D
was in the range of seconds.
Thus, the contribution of T
2D
to the total proton relaxation
was negligible for all studied peat and reference samples.
Consequently, the calculated large T
1
/T
2
ratios of water in
these samples were not due to diffusion in internal magnetic
field gradients, which had to be very small in all studied peat
and reference samples.
The findings for water in agar, allophane gels and swol-
len sago suggest longer correlation times for the dipole
interactions of the bound water protons compared to free
water [25] due to water structuring [17, 23]. Thus, swelling
of these materials reduced both the rotational and
translational water mobility compared to free water, which is
water mobility compared to free water, which is consistent
with the findings for wheat starch pastes [23].
Water State Characterization by
1
H NMR Relaxometry
and DSC
T
1
/T
2
Ratios as a Measure of the Water State in Peat
Samples
Fig. (8) shows the T
1
/T
2
ratios of the rewetted peat soil
samples at the beginning (19C) and end of hydration time
(swollen at 30C, ratios determined at 19C) as a function of
the relative water content (rel. wc). The rewetted peat sam-
ples were measured in a fully water saturated (black) and in a
step-wise desaturated state (grey) after centrifugation (i.e. the
original peat samples from the swelling kinetics experiment
at 30C shown in Fig. 4). The calculation of the T
1
/T
2
ratio
started from 7.5 % rel. wc instead of 0 %, because the num-
ber and position of the peaks at very small T
1
values (Fig.
1A+B, T
1
< 20 ms; Fig. 2A+B, T
1
< 2 ms) were not repro-
ducible for the repetition samples.
The T
1
/T
2
ratios calculated from the step-wise desatu-
rated peat samples were comparable to those from the relaxa-

Fig. (6). Two-dimensional T
1
- T
2
- and T
2
- D - correlation spectra of water in peat 1 (A-C) and peat 2 (D-F) at the beginning (A+D) and
end of hydration time (B+E and C+F) at 30 MHz determined at 20C.

Fig. (7). T1/T2-ratio of water in agar gels as a function of the agar concentration at 7.5 MHz (A) and two-dimensional T
1
- T
2
- (B) and T
2
-
D - correlation spectra of water swollen starchy sago beads (C) at 30 MHz determined at 20C.
A B C
D E F
A B C
D E F
A B C
D E F

0
10
20
30
0 10 20 30
c (Agar) /g L
-1
T
1
/
T
2
A B C
0
10
20
30
0 10 20 30
c (Agar) /g L
-1
T
1
/
T
2
A B C
0
10
20
30
0 10 20 30
c (Agar) /g L
-1
T
1
/
T
2
A B C

tion time distributions of the fully water saturated peat sam-
ples at the beginning and end of hydration (Fig. 8). The only
exception was again RS 2 of peat 2 where the T
1
/T
2
ratios of
the desaturated sample were smaller than those of the fully
saturated one. This was maybe due to the decanting of the
peat samples into the centrifugation tube and the resulting air
contact and re-packing of the sample. The T
1
/T
2
ratios of
water in the peat samples increased with decreasing rel. wc
from 1.1 (at rel. wc = 100 %) to 8 - 9 (peat 1) and 6 - 18
(peat 2) at rel. wc of 10 - 20 %. The amount of water with
T
1
/T
2
ratios larger 3 increased from 40 % at the beginning to
about 60 % (or 80 % for RS 2 of peat 2) at the end of hydra-
tion. The large T
1
/T
2
ratios of RS 2 than RS 1 of peat 2 were
mainly due to the different T
1
distribution (see Fig. 2A).
It was found for both peat samples that at the beginning
(not shown) and end (Fig. 9) of hydration the amount of wa-
ter with T
1
/T
2
ratios larger 3 increased with increasing tem-
perature. Furthermore, the T
1
/T
2
ratios between 30 % and 80
% rel. wc were always larger at 30C than at 5C (Fig. 9).
Eq. 7 was applied to calculate the activation energies, E
A
, of
the temperature dependent increase of the observed T
1
/T
2

ratio increase by using T
1
/T
2
= k . The calculated E
A
at the
beginning and end of hydration was 3 - 12 kJ mol
-1
for both
peat samples. It, therefore, can be suggested that the tem-
perature dependency of the T
1
/T
2
ratios of water in the peat
samples can be attributed mainly to increasing water mobil-
ity (diffusion and rotation) at higher temperatures. Conse-
quently, at least three types of water can be distinguished by
the different T
1
/T
2
ratios. Type 1 is represented by small re-
laxation times and large T
1
/T
2
ratios (> 6 at rel. wc smaller
30-40 % in Fig. 9). Type 3 is represented by long relaxation
times and small T
1
/T
2
ratios between 1.0 and 1.5. Type 2 can
be considered as the transition from type 1 to type 3 and its
amount of water is depending on the mobility of water (see
Fig. 9). We suggest that water type 1 represents bound or
structured water, type 2 exchange water and type 3 bulk-like
free water.
Some of the water in the peat samples was found to be
similar to free pure water over the whole period of hydration
(T
1
/T
2
ratio ~ 1). Another part of the water changed its state
after addition to the peat sample as represented by larger
T
1
/T
2
ratios, which were much larger than generally found in
rock core samples [24]. These large T
1
/T
2
ratios of water in
peat were in the same range as determined for agar gel or
swollen sago beads (Fig. 7). This suggests that this water
was structured by the peat in a comparable way as reported
for water in starch [17]. Consequently, the large T
1
/T
2
ratios
in the peat samples may be due to a water structuring in the
vicinity of the peat surfaces and the resulting longer rota-
tional correlation times of the water molecules within this
water phase [17]. The amount of structured water increased
during hydration, because the amount of water with T
1
/T
2

ratios larger 3 increased by a factor of 1.5. This indicates that
about 20 % of the total water significantly changed its state
during the swelling, which suggests that the amount of water
within a gel phase in the peats increased.
Cryo-NMR Relaxometry: Determination of Non-Freezable
and Loosely Bound Water
The amounts of non-freezable water at -34C of the two
peat samples determined by Cryo-NMR ranged between 0.43
g g
-1
d.m. and 0.55 g g
-1
d.m. and increased by 5 % (peat 1)
and 20 % (peat 2) during hydration (Table 3). From the
freezing behaviour determined by DSC, it can be concluded
that this water was not frozen until -90C (data not shown).
The amounts of loosely bound water at -5C were about 5 to
10 times smaller than those of the non-freezable water at -
34C and increased only for peat 2 during hydration (Table
3). However, the amount of loosely bound water in peat 2
was 20 % to 50 % smaller than for peat 1.
The relaxation time of water in the air dried peat samples
measured at 19C, where only non-freezable water existed,
was 293 3 s for peat 1 and 267 3 s for peat 2. The T
2

values determined by Cryo-NMR represent the relaxation
times of non-freezable water and of loosely bound water in
the frozen peat samples at the maximal water holding capac-
ity. Table 3 shows that the T
2
values for each peat increased
in the following order: non-freezable water in air dried peat,
non-freezable water at -34C and loosely bound water in the

Fig. (8). T1/T2 ratios of the rewetted peat soil samples at the beginning (start) and end of hydration time as a function of the relative water
content (rel. wc) determined from the relaxation time distributions at 19C. The T1/T2 ratios at the end of hydration were calculated from the
30C samples measured at 19C. The rewetted peat samples were measured in a fully water saturated and step-wise desaturated (centrifuga-
tion) state. Relative water contents were calculated with Eq. 4 (saturated samples) and Eq. 5 (desatureated samples). The results of the two
repetition samples or replicates (RS1 and RS2) are displayed.
0 20 40 60 80 100
3
6
9
12
15
18
peat 2 RS 1 RS 2
fully saturated
start
end
desaturated/centrifuge
start
end
B

T
1
/
T
2
rel. wc /%
0 20 40 60 80 100
3
6
9
12
15
18
peat 1 RS 1 RS 2
fully saturated
start
end
desaturated/centrifuge
start
end
A

T
1
/
T
2
rel. wc /%

frozen peat samples. Furthermore, additionally to the in-
creasing amounts of non-freezable water at -34C and of
loosely bound water the T
2
values of both water types in-
creased during hydration.
The amounts of non-freezable water were comparable to
those reported for different starch materials [15] or peats [7],
but were larger than those reported by Schaumann et al. [14].
Increasing amounts of non-freezable water during hydration
were also found for starch gels [52]. The relaxation times of
the two types of bound water were about one order of magni-
tude larger than determined for peat at 300 MHz [7]. The
differences, therefore, may be due to the 10 times higher
magnetic field strengths compared to this study.
DSC: Determination of Different Water States and of
Melting Enthalpy of Freezable Water in Peat
The DSC thermograms in Fig. (10) show the endothermic
melting peaks of free pure water and soil solutions of the two
peat samples extracted at the end of hydration (Fig. 10A+B).
Furthermore, they show the melting peaks of the freezable
water in the fully saturated (Fig. 10C+D) and in the desatu-
rated peat samples after centrifugation at 4000 RPM (Fig.
10E+F) at the beginning and end of hydration time. The
number of melting peaks of water in the fully saturated peat
samples increased from one at the beginning (broad peak
around +2C in Fig. 10C+D) to two peaks at the end of hy-
dration (one sharp peak around 0C and one broad peak be-
tween +2C and +5C). For the desaturated samples, a clear
splitting of the melting peak into one sharp and one broad
peak was observable at the beginning as well as at the end of
hydration (Fig. 10E+F). In contrast to the fully saturated
peat sample, the position of the sharp peak was around -1C
and the width of the broad peak was significantly smaller.
The onset of the melting peak was between -5C and -10C
for both peats. The DSC thermograms of free pure water and
soil solutions consisted of only one melting peak with a very
sharp onset at -3C to -1C and a maximum between +1C
and +2C. For agar gel (30 g L
-1
), the thermogram consisted
of a sharp peak at -0.3C and a broad peak at 5C (not
shown).
Fig. (11) shows that the peak temperature in the desatu-
rated peat samples after centrifugation at 4000 RPM at the
end of hydration time (Fig. 10E+F), agar gel, water saturated
sand 150 m and free pure water increased with increasing

Fig. (9). T1/T2 ratios of the rewetted peat soil samples at the end of hydration time as a function of the relative water content (rel. wc) deter-
mined from the relaxation time distributions of water in the fully saturated peat samples at 5C and 30C. The results of the two repetition
samples or replicates (RS1 and RS2) are displayed.
Table 3. Results of the Cryo-NMR and DSC Measurements of the Two Peats at the Beginning and End of Hydration Time. SD
Standard Deviation, SE Standard Error, n.s. not Significant. T
2
of Water in the Air Dried Samples was 293 3 s for
Peat 1 and 267 3 s for Peat 2. No Enthalpic Peak was Observed in the DSC Thermogram for Both Air Dried Peat
Samples
Cryo-NMR DSC
Non-Freezable Water
at -34C
Loosely Bound Water
at -5C
Non-Freezable
Water
Melting
Enthalpy of
Water in Peat
Enthalpy
Sharp Peak
Water
Content
Sharp Peak
Sample
wc SD
/g g
-1
d.m.
T2 SD
/s
wc SD
/g g
-1
d.m.
T2 SD
/s
wc SE
/g g
-1
d.m.
Hf SD
/J g
-1
Hf SD
/J g
-1
d.m.
wc SD
/g g
-1
d.m.
start 0.524 0.001 435 2 0.110 0.001 925 5 0.85 0.11 366 8 199 38 0.54 0.10 peat 1
end 0.553 0.005 469 5 0.106 0.001 973 26 0.77 0.05 347 7 265 42 0.71 0.11
start 0.428 0.007 402 3 0.058 0.001 794 6 0.54 0.24 329 44 95 11 0.28 0.03 peat 2
end 0.522 0.012 568 83 0.083 0.001 1047 92 0.60 0.08 326 9 158 26 0.49 0.06
0 20 40 60 80 100
3
6
9
12
15
18
peat 2 RS 1 RS 2
end
05C
30C
B

T
1
/
T
2
rel. wc /%
0 20 40 60 80 100
3
6
9
12
15
18
peat 1 RS 1 RS 2
end
05C
30C
A

T
1
/
T
2
rel. wc /%

heating rate. Thus, the melting of freezable water was kineti-
cally controlled in all studied samples. However, the heating
rate dependence was pronounced stronger for the broad peak
than for the sharp peak. Furthermore, the heating rate de-
pendence of the broad peak of the two peats was less pro-
nounced than for agar gel, but stronger than for free pure
water. For peat 1, it was comparable to sand 150 m.
The amounts of non-freezing water determined from
DSC were ~0.8 g g
-1
for peat 1 and ~0.6 g g
-1
for peat 2 and
were comparable for the beginning and the end of hydration
(Table 3). The melting enthalpies of freezable water,
H
f
, in
the peats (Table 3) were in the same range as those of free
pure water and of soil solution 338 8 J g
-1
and decreased
slightly only for peat 1 during hydration. To test whether the
amount of water, represented by the sharp peak in the DSC
thermogram, increased during hydration, the enthalpy of the
sharp peak,
H
f
, (integral between peak onset at low tem-
perature and minimum between the sharp and broad peak
using the same baseline as for the total peak) was calculated
for the peat sample after centrifugation at 4000 RPM. This
made the observation of the sharp peak at the end of hydra-
tion possible for the full saturated peat samples. Assuming a
melting enthalpy independent of the water binding state, we
estimated the water content from
H
f
/ H
f
(
H
f
per gram of
dry peat), of the sharp peak for the beginning and end of
hydration. Table 3 shows that this calculated water content,
represented by the sharp peak, increased for both peat sam-
ples during hydration. However, this increase was significant
only for peat 2, where the water content increased by about

Fig. (10). DSC thermograms of free pure water and soil solutions of the two peat samples extracted at the end of hydration (A+B) and of the
freezable water in the fully saturated (Fig. 10C+D) and in the desaturated peat samples after centrifugation at 4000 RPM (Fig. 10E+F) at the
beginning and end of hydration time. The results of two of the three repetition samples used in the DSC are displayed as black and grey col-
oured curves.
60 %. This water accounts for 3 - 5 % of the total water in
peat 1 and 6 - 11 % in peat 2.
A melting point depression was found for some water in
the peats, which was not caused by dissolved salts in the soil
solution. The melting characteristic of the water was strongly
affected by the peat matrix and resulted in a splitting into (at
least) two to three different types of water represented by the
broad onset at low temperatures and the two peaks in the
DSC thermograms, respectively. The broad shape of the on-
set may be due to the binding forces for water molecules
within this boundary phase, which change strongly with in-
creasing distance from the peat surfaces. At a certain dis-
tance from the surfaces and outside this boundary phase, the
water molecules are still affected by the surfaces but in a
more uniform way. These uniform conditions resulted in the
formation of the sharp melting peak between -2C and 0C.
We thus assume that the sharp peak mainly represents
loosely bound water in peat. The shape of the broad melting
peak of water in the fully saturated peat samples is different
than for free pure water or for soil solution. However, the
temperature range and maxima are comparable to water and
soil solution where no loosely bound water exists (Fig. 10).
Hence, the broad melting peak may represent freezable bulk
water, but additionally water that was still affected by the
peat matrix.
According to the findings for maltodextrin gels [15], it
can be suggested that the melting peak splitting of water in
peat was due to the entrapment of water in a gel phase. By
interpreting the energy transformation related with the sharp
peak as quantitative measure for the amount of water in gels,
the water contents related to the sharp peak at the beginning
and end of hydration (Table 3) indicated the formation of
new and/or the change of existing gel phases during hydra-
tion. This would then indicate that the amount of water in
gels increased during hydration and that some water was
entrapped in gel phases shortly after water addition. This
suggests an initial formation of gel phases in peat after sur-
face wetting.
A splitting of the melting peak of water in peat in the
DSC thermograms was also found by McBrierty et al. [7]
and by Schaumann [14] for peats at water contents below the
maximal water holding capacity. For peats with relatively
high water contents, no splitting of the melting peak was
observed [7]. The melting enthalpies of freezable water and
the amounts of non-freezable water reported by McBrierty et
al. [7] and Schaumann [14] were smaller than the values
calculated in this study. The peak maxima are located at
lower remperatures than reported by McBrierty et al. [7], but
at higher temperatures than those reported by Schaumann
[14]. In the first study, both peak maxima were detected
above 0C, whereas in the latter both peak maxima were
found below 0C. This may be due to the higher heating
rates compared to our study or because different peat sam-
ples were used. Schaumann [14] found a heating rate de-
pendence only for the broad peak and concluded that the
broad peak represents loosely bound water and the sharp
peak bulk-like water, which is contrary to the conclusions of
this study. These differences may be due to the much higher
degradation state and the lower water contents of 0.4 - 0.6 g
g
-1
of the peats used by Schaumann [14] and due to the dif-
ferent experimental setups (hydration from the gas phase,
higher heating rates). For gelatine gels, Liu and Yao [57]
attributed the sharp peak to loosely bound water (referred as
intermediate water) and the broad peak to free water.
Comparison of the Results Determined by
1
H NMR and
Cryo NMR Relaxometry and DSC
The amounts of non-freezable water determined by DSC
were larger than those from Cryo-NMR, but were in the
range of the sum of non-freezable and loosely bound water
determined by Cryo-NMR (Table 3). 3 - 5 % of the total
water and 10 - 13 % were determined as non-freezable water
in peat 1 and peat 2, respectively. The value for peat 2 agrees
roughly with the beginning of the plateau at large T
1
/T
2
ra-
tios observed in Fig. (8B). For peat 1, no T
1
/T
2
ratio was
calculated at this low relative water content (see above). The
sum of non-freezable water and water represented by the
sharp peak in the DSC thermogram accounts for 8 - 10 % of
the total water in peat 1. This value agrees with the largest
T
1
/T
2
ratio in Fig. (8A). In peat 2, this sum of the two water
types accounts for 17 - 25 % of the total water, which agrees
with the end of the plateau with large T
1
/T
2
ratios in Fig.
(8B). This suggests that the large T
1
/T
2
ratios of water in the
peat samples were related to non-freezable water and water
represented by the sharp peak in the DSC thermograms
(loosely bound water and water in gels). Thus, the observed

Fig. (11). Maxima of the DSC melting peaks of the desaturated peat samples after centrifugation at 4000 RPM at the end of hydration time
(Fig. 10E+F), agar gel (30 g L
-1
), water saturated sand (particle size 150 m) and free pure water as a function of heating rate.
0 2 4 6 8 10
-1
0
1
2
3
6
9 broad peak maximum
peat 1
peat 2
agar gel
sand 150 m
water
B
t
e
m
p
e
r
a
t
u
r
e

/
C
heating rate /K min
-1
0 2 4 6 8 10
-1
0
1
2
3
6
9
A

sharp peak maximum
peat 1
peat 2
agar gel
t
e
m
p
e
r
a
t
u
r
e

/
C
heating rate /K min
-1

changes in the T
1
/T
2
ratios during hydration may be due to
the increasing of amounts of these two water types.
From the water state characterization by
1
H NMR and
Cryo NMR relaxometry and DSC we conclude that a layered
bound water phase existed inside the peats. This phase con-
sisting of several layers of non-freezable and loosely bound
water, whereas the binding forces and, consequently, the
water structuring decreases with increasing distance from the
peat surfaces resulting in higher mobility of the water mole-
cules. This layered bound water phase includes water en-
trapped in gel phases. During swelling, the amounts of
bound water as well as the mobility of the water molecules
inside the bound water phase increased, which indicates a
water intrusion into the peat matrix as well as a reorientation
of SOM chains into the pore space. This is consistent with
the conclusions drawn for mineral soil samples [6] and peat
samples [8]. Mikutta et al. [58] studied the hydration of po-
lygalacturonate (PGA) coatings on alumina (Al
2
O
3
) and con-
cluded from their NMR and DSC results that the PGA chains
reorient into the pore space upon swelling.
Microbial Activity
Throughout the 21 days incubation the microbial activity
in the investigated samples at 60 % of the maximal water
holding capacity was constant and extremely low. For peat 1
it varied from 0.06 to 0.07 mg CO
2
h
-1
100 g
-1
and for peat 2
from 0.08 to 0.1 mg CO
2
h
-1
100 g
-1
of soil. Additional repli-
cates of the peat samples that were fully saturated showed
almost no release of CO
2
during incubation. The values of
the CO
2
release from the two peats were about 100 to 1000
times smaller than reported for two mineral soil samples,
where relaxation time distribution changes were significantly
stronger in samples with high microbial respiratory activity
[13]. Consequently, the microbial effects on the changes of
relaxation time distribution of water in the two peat samples
during hydration may be considered negligible.
Synthesis
The changes of the relaxation time distributions of water
in the two peat samples as well as the increase of the Q val-
ues during hydration were most probably not caused by in-
fluences from the soil solution, internal magnetic field gradi-
ents, surface relaxivity changes or soil microorganisms and,
therefore, indicate swelling of the two studied peat samples
[1, 2, 6, 12, 13]. The swelling of peat was characterised by
redistribution of water, by increasing amounts of non-
freezable and loosely bound water, by the formation of gel
phases as well as by the reduction of the translational and
rotational mobility of water molecules in the two peat sam-
ples. Furthermore, swelling induced strong changes of the
pore size distributions, which resulted in the reduction of
number of large pores (> 50 m) and formation of medium-
sized pores (50 - 10 m). Some time after two days of hydra-
tion the formation of small-sized pores (< 10 m) was also
observed.
It was found that the stronger volumetric swelling of peat
2 was linked to stronger changes of the pore size distribution
and higher amounts of redistributed water as well as to
stronger relative increases of the amounts of non-freezable
and loosely bound water. In addition, more water was af-
fected by the reduction of the translational mobility of water
molecules. We suggest that this was due to the higher degra-
dation state and the more heterogeneous matrix of peat 2. It
is very likely that air drying of peat 2, previous to the re-
hydration, caused stronger matrix changes, such as closing of
small-sized pores and collapsing of gel phases [6, 59].
The physical property of the volumetric swelling was
connected to various changes of physical and physicochemi-
cal properties of peat during hydration. Stronger volumetric
swelling was accompanied by stronger changes of physical
and physicochemical properties of peat. The peat swelling
was governed by three processes with time constants in the
range of minutes (fast process), hours (medium fast process)
and weeks/months (slow process) with related apparent acti-
vation energies of 5 - 50 kJ mol
-1
, indicating the breaking of
hydrogen bonds [53], water diffusion and reorientation of
SOM chains during hydration [6, 38, 49].
CONCLUSIONS
The increasing amounts of non-freezable and loosely
bound water as well as the reduction of the translational and
rotational mobility of water molecules in the two peat sam-
ples indicate changes of the physical and physicochemical
properties of the peats during swelling at constant tempera-
ture and moisture conditions. These changes took place over
three time periods between minutes and months and were
governed by physical and physicochemical processes. From
the results we derived a mechanistic model to describe the
fundamental processes of peat swelling. This model is based
on results obtained from air dried peat samples with low wa-
ter contents. This water exists as non-freezable water in peat.
Within the first minutes after wetting of the initially water-
accessible peat surfaces water structuring and water reorien-
tation in the vicinity of the surfaces took place. This resulted
in the increase of the existing non-freezable water phase due
to the addition of new layers of structured water. Parallel to
this process the formation of loosely bound water and gel
phases occurred. Within the next following 20 to 50 hours,
the swelling was mainly controlled by the diffusion of water
into the peat matrix, which results in volumetric swelling and
the formation of medium-sized pores (> 10 m). After this, a
very slow reorientation of SOM chains together with an on-
going volumetric swelling took place, which lasted for sev-
eral months. During this time formation of medium-sized
pores (> 10 m) as well as small-sized pores (< 10 m) oc-
curred. Parallel to the processes which were observed during
the last two time periods, intra-particulate surfaces became
water-accessible which in turn resulted in the formation of
non-freezable, loosely bound and gel water. The findings of
this study are of environmental importance for helping to
optimise renaturation and rewatering of commercially used
peatlands and to better understand sorption/desorption and
transport processes of pollutants and nutrients in natural or-
ganic matter rich soils.
ACKNOWLEDGEMENTS
This study was funded by the German Research Founda-
tion, DFG, (SCH849/5-3). The T
1
/T
2
- and T
2
/Diffusion-
correlation measurements were supported by the European
Community activity Large-Scale Facility Wageningen NMR
Center (FP6-2004-026164, 2006-2009). We like to thank Dr.
Song from Schlumberger for the ILT programme. A special
thank you goes to Julia Bayer (University Koblenz-
Landau) for her help in improving the manuscript.
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Received: July 26, 2009 Revised: November 30, 2009 Accepted: December 04, 2009

Jaeger et al.; Licensee Bentham Open.

46 The Open Magnetic Resonance Journal, 2010, 3, 46-51

1874-7698/10 2010 Bentham Open
Open Access
Investigation of Iron(III)-Release in the Pore Water of Natural Sands by
NMR Relaxometry
Ivonne Mitreiter*
,1,2
, Sascha E. Oswald
2,3
and Frank Stallmach
1

1
University of Leipzig, Department of Interface Physics, Linnstr. 5, 04103 Leipzig, Germany
2
UFZHelmholtz Centre for Environmental Research, Department of Hydrogeology, Permoserstr. 15, 04318 Leipzig,
Germany
3
University of Potsdam, Institute of Earth and Environmental Science, Karl-Liebknecht-Strae 24-25, 14476 Potsdam,
Germany
Abstract: Proton nuclear magnetic resonance (NMR) relaxometry offers a non-invasive and non-destructive measurement
method to observe and to visualise changes in the iron(III)-ion concentration in aqueous solutions. This is possible due to
its paramagnetic influence on the relaxation times. In the context of mineral dissolution processes in natural sediments, the
effect of the presence of dissolved iron(III) on the NMR relaxation times of the pore water was investigated. The decrease
in the relaxation times T
1
and T
2
corresponding to an increase in the dissolved iron(III) concentration was quantified. This
relation was used to monitor relative changes in the concentration of dissolved iron(III)-ions in natural sands.
Experiments were conducted to calibrate the iron(III) concentration from the measured relaxation times. These were done
in bulk water as well as in iron(III)-mineral bearing sands to take the effect of surface relaxation into account. It was
shown that for relatively coarse-grained sand fractions the effect of iron(III)-ions in solution dominates. This allows the
determination of the dissolved iron(III) concentration in natural sands by NMR. The method also enables us to capture the
changes in the iron(III) concentration with high temporal resolution. This was demonstrated in column experiments, in
which an acid (hydrochloric or sulphuric acid) was applied from the top on the sands to dissolve the mineralogical bound
iron(III) and were the dissolution of iron(III) can be captured with sufficient temporal resolution.
Keywords: Paramagnetic ion, iron(III), relaxation, mineral dissolution.
INTRODUCTION
Acid mine drainage is globally a major environmental is-
sue. The groundwater is often heavily polluted near mines of
sulphide minerals. This is due to the continuously release of
low pH and heavy-metal loaded water from the mines drain-
ing into groundwater [1]. The oxidation of pyrite and other
metal-sulphide minerals by oxygen plays a key-role in acid
mine drainage. It acts as a source of sulphate and iron (Fe) in
groundwater, and of heavy metals in general in the environ-
ment [1]. Dissolved iron is not detrimental to human health,
but high concentrations have a negative impact on the use-
fulness of the water. It can cause clogging of well-screen
openings and pumps and has an unpleasant metallic taste.
Iron exists either in a ferrous Fe
2+
or ferric Fe
3+
state. In
which form iron is dissolved in water depends on the amount
of oxygen and upon its degree of acidity. Fe
2+
is oxidised to
Fe
3+
in contact with oxygen or by the action of iron related
bacteria. Fe
2+
-ion is very soluble, but Fe
3+
-ion is only soluble
at low pH values in water. Iron is a major component in acid
mine waters. Precise measurements are important in order to
understand the processes taking place regarding the dissolu-
tion of Fe
3+
-ions.

*Address correspondence to this author at the UFZHelmholtz Centre for
Environmental Research, Department of Hydrogeology, Permoserstr. 15,
04318 Leipzig, Germany; Tel: +493412351253; Fax: +493412351837;
E-mail: ivonne.mitreiter@ufz.de
In most present analytical methods [2], Fe
3+
-ion is deter-
mined by computing the difference between the total dis-
solved Fe and the dissolved Fe
2+
, i.e. Fe
3+
-ion concentrations
are determined in an indirect way. The determination of
Fe
3+
-ion concentration in water is based on the determination
of Fe
2+
-ion concentrations, followed by a separate determina-
tion of the total dissolved Fe concentration after the reduc-
tion of Fe
2+
. The difference between the concentrations of
total dissolved Fe and Fe
2+
is taken as the Fe
3+
-ion concen-
tration. One major problem with this approach is the overes-
timation of Fe
3+
-ion concentration at high Fe
2+
-ion concen-
trations in the analyzed sample, where the difference be-
tween total dissolved Fe and Fe
2+
is comparable to the error
of the determination.
In this study we applied nuclear magnetic resonance
(NMR) relaxometry measurements as a method for the direct
determination of dissolved Fe
3+
-ion concentrations. This is
possible due to the paramagnetic properties of Fe
3+
-ions in-
fluencing the NMR measurements. Mostly, paramagnetic
ions (e.g. Fe
3+
, Mn
2+
, Ni
2+
and Cu
2+
) are seen as a complicat-
ing factor in the NMR interpretation due to their significant
influence on the relaxation times. Previous studies concen-
trate on the effects of solid paramagnetic species and par-
amagnetic ions adsorbed to the solid phases [3, 4]. In many
natural samples the iron concentrations are high enough to
have a significant influence on the surface relaxivity. Bryar
et al. [5] point out to be careful with the interpretation of
Investigation of Iron(III)-Release in the Pore Water The Open Magnetic Resonance Journal, 2010, Volume 3 47
pore size distributions between samples unless the iron con-
centration is observed to be the same. Bryar et al. [5, 6]
showed that the relaxation rates of Fe
3+
-solutions depend
linearly on the Fe
3+
-ion concentration.
Present NMR techniques allow a wide range of applica-
tions. They can be used to provide information on the tempo-
ral and spatial distribution of water and dissolved ions, on
flow and transport processes [7-10]. NMR has also been
used to investigate several microbial processes and biofilm
properties via relaxation time differences [11,12]. NMR ap-
plications are used to characterize sediments [13-16] and in
well logging [17].
The aim of this study is to apply NMR relaxometry
measurements to determine directly the dissolved Fe
3+
-ion
concentrations in the sediment pore water. By measuring the
relaxation times of water saturated sediment samples and
taking surface relaxation at the pore matrix interface into
account, the Fe
3+
-ion concentration in the pore water solution
can be calculated. Additionally, column experiments are
performed which show that this approach allows to monitor
the time dependence of the dissolution of naturally occurring
Fe
3+
in minerals. Thus, NMR relaxometry is used to gain
non-invasive and non-destructive insights into such proc-
esses with high temporal resolution.
BACKGROUND AND THEORY
Relaxation in Solution and in Porous Media
The relaxation times of water decrease with increasing
viscosity and increasing amount of solvents, respectively,
and especially with increasing amount of dissolved par-
amagnetic species, such as dissolved oxygen or Fe
3+
-ions.
The theoretical background to these relaxation processes in
liquids was founded by the Bloembergen-Purcell-Pound
(BPP) theory [18]. Small concentrations of dissolved par-
amagnetic metal ions cause huge decrease of the relaxation
times. Hence, the bulk relaxation rate is a sum of the par-
amagnetic and diamagnetic contributions and consequently
proportional to paramagnetic ion concentration as well as the
number of water molecules in the hydration sphere of an ion
[19, 20]. The relaxation rates in bulk water (1/T
1b
, 1/T
2b
)
with dissolved paramagnetic ions depend linearly on the ion-
concentration c:
1/ T1b, 2b =1/ T1b0, 2b0 + R1, 2 *c (1)
where T
1b0
and T
2b0
are the relaxation times of paramagnetic
ion-free water. The relaxivities R
1,2
of the paramagnetic ion
depends on the electron spin state of the ion and the mag-
netic field strength at which the measurement is performed.
The relaxation rate of water in a porous material is larger
than in bulk water because of additional mechanisms, which
enhance the relaxation. The restriction of the water mole-
cules at the solid pore walls of the pore space and the content
of paramagnetic ions at the surface of these particles cause
an additional surface relaxation mechanism [21]. So the lon-
gitudinal and transverse relaxation rates can be described as
a sum of relaxation rates:
1 / T1, 2 = 1 / T1b, 2b +1 / T1s, 2s (2)
where T
1,2b
and T
1,2s
are the bulk and surface relaxation
times. In the fast diffusion regime (meaning that all protons
travel to and relax at the pore surfaces in the time interval of
the NMR experiment) the surface relaxation time depends on
the volume-to-surface ratio V/S of the pores and is given by
T 2s = 1/ s *V / S (3)
where
s
is the surface relaxivity. It is influenced by the in-
teraction of the pore fluid molecules with the internal pore
surface and has been found to increase with the concentra-
tion of minerals containing paramagnetic ions on the pore
surface [22].
NMR Pulse Sequences
By applying a series of RF pulses, the total nuclear mag-
netization of the
1
H nuclei of the (pore) water (i.e. the spin
system) can be manipulated. Such pulse sequences can be
used to determine the relaxation times. The following se-
quences have been implemented in the work reported in this
paper: inversion recovery (IR) sequence [23] and the Carr-
Purcell-Meiboom-Gill (CPMG) echo train [24, 25]. The IR
sequence was used for T
1
measurements. In this sequence an
inverting RF pulse is followed, after the time interval t, by
a /2 pulse (Fig. 1a). During the time interval t, the mag-
netization is subjected to longitudinal (T
1
) relaxation only.
The CPMG sequence was used to obtain T
2
. After an initial
excitation of the magnetization by a /2 RF pulse a spin echo
train is generated by RF pulses (Fig. 1b).
Paramagnetic Iron(III)-Ions
Paramagnetic ions are found to have a strong influence
on the NMR relaxation times. A paramagnetic species com-
monly found in groundwater, soils and sediments is Fe
3+
.
Fe
3+
-ions have the electron spin quantum number S = 2.5.
Compared to this, Fe
2+
-ions have a lower electron spin val-
ues of 2. Due to this lower electron spin quantum number
Fe
2+
-ions have a smaller effect on the relaxation times.
Hence NMR measurements allow the observation of redox
reactions using the Fe
3+
- Fe
2+
-redox pair [6]. In another
chemical form, i.e. as complexed Fe
2+
, iron has recently been
used to characterize biofilms [26].
The most common iron-oxides are goethite (-FeOOH),
hematite (-Fe
2
O
3
), lepidocrocite (-FeOOH), ferrihydrite
(Fe(OH)
3
*nH
2
O) and magnetite (Fe
3
O
4
). These iron-oxides

Fig. (1). Pulse sequences used in this study, a) IR sequence to obtain T
1
, b) CPMG for measuring T
2
.

48 The Open Magnetic Resonance Journal, 2010, Volume 3 Mitreiter et al.
are pure Fe
3+
-minerals, with the exception of magnetite
which contains both Fe
3+
and Fe
2+
. The concentration of total
iron in soils and sediments is normally around 0.2-5%. For
porous media Foley et al. [3] and Bryar et al. [5] have shown
that the surface relaxivity
s
and with this the surface relaxa-
tion rate 1/T
1,2s
is linearly proportional to the concentration
of paramagnetic ion containing minerals on the pore surface.
In groundwater and soil water, iron exists either in a fer-
rous Fe
2+
or ferric Fe
3+
state. Which form iron takes, is con-
trolled by pH and redox potential. Ferric iron is relatively
insoluble in water. Only at pH values below 3.0 it is ex-
pected to appear in solution, e.g. in acidic water from metal
mines and acidic forests soils. The relaxation rates of Fe
3+
-
solutions are sensitive to pH-dependent speciation of the ion
[5]. In aqueous solution at pH 1.0, Fe
3+
is present mostly
complexed by six molecules of water, [Fe(H
2
O)
6
]
3+
, but at
pH 3.0 hydrolysis reactions change the iron to a mixture of
[Fe(H
2
O)
6
]
3+
, [Fe(H
2
O)
5
OH]
2+
and [Fe(H
2
O)
4
(OH)
2
]
+
[5].
The number of exchangeable water molecules in the hydra-
tion sphere decreases as pH increases [5].
With increasing pH, the Fe
3+
adsorbs to the solid surfaces
of surrounding porous media. Bryar et al. [5] showed that at
pH 3.0 roughly 20 % of Fe
3+
can be adsorbed to surfaces.
The presence of adsorbed Fe
3+
ions on the surface as well as
Fe
3+
-bearing solid phases present as surface coatings or as
separate mineral grains also significantly increase the surface
relaxation.
MATERIALS AND METHODS
Materials and NMR Sample Preparation
The sand samples investigated originate from a sand de-
posit about 10 km west of Leipzig, Germany. It was formed
as a terminal moraine during the ice ages of the middle Pleis-
tocene. Previous analyzes of sand samples from the same
location [16] showed that the sand consists of quartz and
feldspar and that the averaged elemental composition of the
grain surfaces contains iron (Al
1
Si
2.3
O
9.3
Fe
0.4
(Mg,Ca)
0.2
(Na,K)
0.1
). In the lab the sand was air dried and sieved yield-
ing 5 fractions with grain diameters in the following ranges:
63 m 125 m 200 m 500 m 800 m 1 mm.
The fluids used in this study were distilled water and so-
lutions of Fe
3+
. The iron solutions were prepared in distilled
water using ferric chloride (FeCl
3
* 6 H
2
O) with the pH ad-
justed at pH 1.0 using hydrochloric acid. Although the pH of
pore fluids in natural settings will not often be as low as
those used in this experiment, we used solutions with high
acidity to ensure a complete dissolution of Fe
3+
-ions, which
facilitates the interpretation of the results. Measurements of
the relaxation time of the bulk fluids (T
1,2b
) were made using
approximately 2 ml of the equilibrated fluid in an NMR tube.
The water-saturated sands were prepared by mixing 6 g
sand with 1.5 ml distilled water in acid-washed glass tubes.
Then acid (hydrochloric acid, sulphuric acid) was added
from the top. For observing the time depended reactions with
acid the measurements started right after sample preparation.
All other sand samples prepared with an acid were left for
seven days to insure the completeness of the reaction. Before
NMR data were collected, excess fluid was removed from
the surface of the sample. NMR data for each sample were
collected at room temperature (22C). Evaporative losses of
water from the sample during data collection were avoided.
NMR Experiments
The NMR relaxometry measurements were performed
using a PC-controlled NMR console MARAN DRX (Reso-
nance Instruments, GB). The home-built permanent magnet
permits a magnetic flux density of B
0
= 0.2 T, corresponding
to a
1
H resonance frequency of 9.1 MHz. The NMR samples
have 20 mm outer diameter and the same filling height. In
the pulse sequences the length of the /2 and rf pulses was
7 and 14 s. The T
1
-weighted time interval t of the IR pulse
sequence was varied from 5 to 5,000 ms. In order to provide
a sufficiently high signal-to-noise ratio, a minimum of 16
scans were acquired for each t-value. The inter-echo time
in the CPMG pulse sequence was varied from 100 to 400 s.
A repetition delay time of RD = 5 s was sufficient to allow
the nuclear spins to relax to equilibrium after each individual
scan.
Fe
3+
-Ions in Solution
We measured the longitudinal and transverse relaxation
rates (T
1
-1
and T
2
-1
) of solutions containing Fe
3+
- ions with
concentrations ranging from 0 g l
-1
up to 5 g l
-1
at a pH of
1.0. Both measured relaxation rates are shown as a function
of the concentration of dissolved Fe
3+
-ions in Fig. (2). The
relaxation rates increase linearly with increasing Fe
3+
-ion
concentration, which corresponds well with the expectation
according to the BBP theory. Linear fits for both relaxation
rates were generated and are presented in Fig. (2) as solid
(T
1
-1
) and dashed lines (T
2
-1
), respectively.

Fig. (2). The dependence of the bulk water relaxation rates T
1b
-1
and
T
2b
-1
on dissolved Fe
3+
-ion concentration at pH 1.0 and room tem-
perature (22C). The solid line in the graph is the calibration plot
for T
1
-1
, the dashed line is the one for T
2
-1
. Please note the logarith-
mic scale, which contort the linear dependency, especially for Fe
3+
-
ion concentrations below 10 mg l
-1
.

From the slope data of these calibration plots, using
Equation 1, the relaxivities of Fe
3+
-ions in water, R
1
and R
2
,
were determined to be R
1
= 0.1792 0,0040 s
-1
mg
-1
l and R
2

= 0.1393 0,0018 s
-1
mg
-1
l (with correlation coefficients R
2

> 0.997).

These results are used to calibrate relaxation rates meas-
ured by NMR to Fe
3+
-ion concentrations. In the following
experiments we measured one of the two relaxation rates and
then calculated the Fe
3+
-ion concentration in solution. This
allows us to determine the Fe
3+
-ion concentration in solution,
without the necessity of destroying the sample or use any
other analytical tool. Therefore, this technique can be used to
capture the temporal progress of such dissolution reactions.
Relaxation Times T
1,2
in Water-Saturated Sands
Internal surfaces of porous media have a significant in-
fluence on the relaxation times. To assess this influence for
the samples of natural sands the relaxation times of distilled
water in five size fractions of the sand were measured. The
results are shown as a function of the grain diameter in Fig.
(3). Relaxation times in the sands are reduced compared to
the relaxation times of bulk water. The smaller the grain
sizes the larger is the decrease of the relaxation times. This is
due to the interaction of the water
1
H nuclei with the surface
of the sand grains. This effect on relaxation time is repre-
sented by the
s
*S/V term in Equation 2.

Fig. (3). Relaxation times T
1
and T
2
of bulk water for five sand
fractions. The diameter d is the mean value of the particular sand
fraction.

For the calculation of the surface relaxivity
s
of water in
the sands a porosity of 0.35 is assumed. The S/V ratio of a
random packing of spherical grains of diameter d is given as
[27].
S / V = 6 *(1 / 1) *1 / d (4)
By substitution Equations 3 and 4 in Equation 2 results
1 / T = 1 / Tb + s * 6 *(1 / 1) *1 / d (5)
With this Equation the surface relaxivity
s
of the sands
are calculated to be 0.0056 cm s
-1
for T
1
-1
and 0.0123 cm s
-1

for T
2
-1
.
Relaxation Times of Water-Saturated Sand with Dis-
solved Fe
3+
-Ions
By adding an acid to a water-saturated sand sample, the
Fe
3+
-containing minerals dissolve and the Fe
3+
-ion concen-
tration in solution increases. Fig. (4) shows the longitudinal
relaxation time T
1
for the different sand fractions without
acid (from Fig. 3, black dots) and after adding H
2
SO
4
(empty
triangles). The addition of the acid (33mol H
+
) to the sand
caused a decrease in the T
1
relaxation time corresponding to
an increase in the Fe
3+
-ion concentration in solution. For the
two fine-grained fractions just small changes in the relaxa-
tion times could be detected because relaxation is fast al-
ready without Fe
3+
-ions. This does not imply that no Fe
3+

was dissolved, rather demonstrates the fact that for these
fractions the surface relaxation is dominant. The larger the
grain size the larger is the relative effect of the dissolved
Fe
3+
-ions on the total relaxation times.

Fig. (4). Relaxation times T
1
for the 5 sand fractions after adding
H
2
SO
4
.

After 7 days time the reaction has presumably ended and
the resulting relaxation times T
1
are in the range from 120
ms to 180 ms for the five sand fractions (Fig. 4). Applying
Equations 1, 2 and 3, the Fe
3+
-ion concentration can be esti-
mated using the following Equation
1/ T1 = 1/ T1b0 + s *S / V + R1 *c (6)
This corresponds to a Fe
3+
-ion concentration ranging
from 3 mg l
-1
for the smallest sand fraction up to 33 mg l
-1

for the largest grain sizes, as seen in Fig. (5). This range is
due to the varying surface effects in the different sand frac-
tions. The error bars were estimated using the Gaussian error
propagation based on the diameter range in each fraction, i.e.
the smallest und the largest diameter in each fraction. It is
clearly seen that for smaller fraction the error in determining
the Fe
3+
-ion concentration is almost as large as the estimated
value. This implies that the estimated Fe
3+
-ion concentration
for the smaller sand fraction is uncertain and therefore, may
be misleading. The grain size used in the following experi-
ment is the fraction from 200 to 500 m, which is large
enough to ensure that the influence of the surface relaxivity
on T
1
and T
2
is low compared to the influence of the Fe
3+
-
ions in solution. Another reason for this choice is the fact
that this fraction of grain sizes has the largest percentage in
our sand sample and generally speaking, this is one of the
most important fractions when considering sand and soil
samples.
As a next step column experiments were performed,
where different concentrations of hydrochloric acid (HCl)
were added from the top to one water-saturated grain size
fraction to dissolve different amounts of Fe
3+
. The added
acid concentrations ranged from 4 mol up to 52 mol H
+
.

50 The Open Magnetic Resonance Journal, 2010, Volume 3 Mitreiter et al.
Starting from the smallest concentration, the acid concentra-
tion was increased in 6 steps till reaching the largest concen-
tration. To investigate effects on changes of surface relaxiv-
ities due to dissolution of mineral phases by increasing acid-
ity the relaxation time distributions were analyzed. To the
sand with the highest acid concentration (52 mol H
+
) so-
dium hydroxide (NaOH) as base was added in surplus. This
leads to the almost complete precipitation of the dissolved
Fe
3+
. The corresponding relaxation time distribution is
dominated by one peak at short relaxation times. In contrast,
the relaxation time distributions before acid addition and
after base addition were found to be very similar. Both
showed a bimodal distribution with much longer relaxation
times than in the state after hydrochloric acid addition and
Fe
3+
dissolution (data not shown). Although after base addi-
tion, the iron precipitates not in the same chemical state and
physical form as it previously existed, the initial relaxation
time distribution can be reproduced, which implies that the
surface relaxivity is not substantially affected by the iron
minerals present.
For observing the dissolution of Fe
3+
-ions with high tem-
poral resolution, the directly measured T
2
relaxation time is
much better suited than the slower T
1
measurement. One T
2
-
measurement by the CPMG-method lasts only a few minutes
while a quantitative T
1
measurement by the IR method re-
quires approximately 15 min up to about one hour (depend-
ing on the required t intervals to capture the relaxation
curve, see Fig. 1). Furthermore, the measurements were per-
formed at a magnetic field of B
0
= 0.2 T, so that influences
on the T
2
relaxation time due to internal field gradients can
be neglected. Thus, we measured T
2
and calculated the Fe
3+
-
ion concentration using the calibration described by the
Equation
c = (1 / T 2 1 / T 2b 1 / T 2s) / R2 (7)
where 1/T
2
is the measured relaxation rate and the term
(1/T
2b
+1/T
2s
), which describes the total relaxation rate due to
bulk and surface relaxation, is determined experimentally
before the addition of the acid. R
2
is known from the slope of
the calibration plot described before (compare Fig. 2). At the
beginning, measurements were taken continuously in time
intervals of minutes. With time passing, i.e. the reaction
slowing down, the time intervals were set larger, up to one
hour between two measurements. The duration of the ex-
periment was one day (24 hours). The temporal progress can
be seen in Fig. (6) for three different acid concentrations.
During the first hours a rapid increase in the Fe
3+
-ion con-
centration in solution is observed, later the Fe
3+
-ion concen-
tration converges towards an asymptotic value. These data
show that the dissolution of Fe
3+
-ions from the minerals is a
fast process. The controlling mechanism to achieve equilib-
rium in the sample is the diffusion of Fe
3+
- ions as well as
H
+
-ions.

Fig. (6). Fe
3+
-ion concentration in the 200 500 m sand fraction
after adding an acid during the first day of reaction.

SUMMARY AND CONCLUSION
The paramagnetic behaviour of Fe
3+
-ions was used in this
NMR relaxometry study to monitor temporal changes in
Fe
3+
-ion concentration in aqueous solution. To derive the
relation between both relaxation times (T
1
and T
2
) and the
Fe
3+
-ion concentration in solution, different solutions were
prepared and the corresponding relaxation times were meas-
ured. A linear relationship was found and parameterised. For
the subsequent experiments the measured relaxation times
could be transformed into Fe
3+
-ion concentration using the
calibration plots. In the next step the influence of the sand
grain size on relaxivities was investigated. As it is well
known from theory, the smaller the particle size the more
dominant is the surface relaxation process. This behaviour
was reproduced for four different sand fractions. The conclu-
sions are that a clear separation between both relaxation
mechanisms, via Fe
3+
-ions in solution and via surface relaxa-
tion, is not possible for fine-grained sands, where the relaxa-
tion times are dominated by the surface relaxation. However,
for larger size fraction of natural sand grains, the NMR
method was shown to be capable to yield the Fe
3+
-ion con-
centration in column experiments, were the increasing dis-
solved Fe
3+
concentrations resulted from the penetration of
an acid front through a sand sample. The addition of acid to
the sample made Fe
3+
-ions dissolve from the iron minerals
present in the sands. The dissolution process was observed at
a high temporal resolution and is characterized by a fast dis-

Fig. (5). The estimated Fe
3+
-ion concentration for each sand frac-
tion with the error bars based on the diameter range for each frac-
tion.

solution of Fe
3+
-ions and diffusion of H
+
and Fe
3+
-ions
through the pores of the sand.
This quantitative approach offers to monitor changes in
dissolved Fe
3+
-ion concentrations due to changes of acidity
in sediments and other release or consumption processes
affecting dissolved Fe
3+
-ions. The experiments proof that the
approach is applicable to natural sediments provided the
grain size is not smaller than about 100 m. Via T
2
meas-
urements the temporal resolution is high enough to follow
changes in concentration induced by geochemical and also
microbial turn-over processes. Doing so, it should be noted
that the formation of metal-organic complexes in sediment-
and soil-solutions can reduce the relaxivities of Fe
3+
as com-
pared to those in model solutions [28]. Besides the dissolu-
tion of paramagnetic ions the swelling of soil organic matter
and the production and release of extracellular polymeric
substances (EPS) by microbes can lead to changes in spin
relaxation mechanisms and should be considered [29].
Methodological conclusions of the presented study are
that for natural sands of medium and coarse size fractions the
dissolution of Fe
3+
-ions from natural, Fe
3+
-containing miner-
als causes a substantial decrease of both, T
1
and T
2
relaxation
times. Future use of the presented approach could make use
of systems with low-field permanent magnets, in our case
providing B
0
= 0.2 T, which are cost-effective or even mo-
bile. In general, a number of situations can be assessed in
terms of changes in dissolved Fe
3+
, for example (i) rate of
acidification and leaching in mine heaps, (ii) the dissolution
of Fe
3+
in soil induced by organic acids produced by plant
roots to improve iron uptake from soil or (iii) iron reduction
and re-oxidation during microbial degradation of organic
contaminants in aquifers and Fe
3+
precipitation from solution
in general.
ACKNOWLEDGEMENTS
Financial support by the DFG (Germany) and the NWO
(The Netherlands) via their joint International Research
Training Group Diffusion in Porous Materials is gratefully
acknowledged. The authors thank Dr. J. Kolander (Univer-
sity of Leipzig, Germany) for his assistance with NMR
measurements.
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Received: May 11, 2009 Revised: December 01, 2009 Accepted: January 26, 2010

Mitreiter et al.; Licensee Bentham Open.


1874-7698/10 2010 Bentham Open
Open Access
A Possible Difference in the Surface Relaxivity of Costal and Inland Sands
Joseph P. Hornak*
,1
, Gianni Ferrante
2
, Andrew Coy
3
and Evan R. McCarney
3

1
Rochester Institute of Technology, Rochester, New York, USA;
2
Stelar, Mede (PV), Italy;
3
Magritek, Wellington, New
Zealand
Abstract: The
1
H nuclear magnetic resonance (NMR) spin-lattice relaxation rate of hydrated sands is related to the sur-
face-to-volume ratio of the voids or pores between the hydrated sand grains and the surface relaxivity of the grains. The
electron spin resonance (ESR) signal is often used to predict the relative surface relaxivity as the surface relaxivity is
thought to be proportional to the concentration of paramagnetic species in the sand grains. We have identified a discrep-
ancy in the surface relaxivity and ESR signal of an ocean beach sand compared to two sands of similar diameter from in-
land deposits. This difference can either be due to more surface weathering of the inland sand or more paramagnetic mate-
rial from seawater adhering to the ocean sand.
Keywords: Hydrated sand, NMR surface relaxivity, spin-lattice relaxation rate, R
1
.
INTRODUCTION
The relationship between the
1
H nuclear magnetic reso-
nance spin-lattice relaxation rate (R
1
) of a hydrated porous
material, the R
1
of bulk water filling the pores (R
1B
), the sur-
face relaxivity of the pores (), and the surface to volume
ratio of the pores (S/V)
P
is the basis of many geophysical
studies related to soil [1-3].
R
1
= R
1B
+ (S/V)
P
(1)
NMR spin relaxation values are used to study hydration,
porosity, and soil contamination [4-6]. Magnetic resonance
sounding [7] is useful for finding aquifers and the R
1
value is
often used to infer pore size. Near-surface magnetic reso-
nance imaging [8,9] of hydrated soil may one day allow im-
aging of buried utilities. The signal from this technique is
strongly dependent on the R
1
value of the hydrated soil.
We recently reported on this relationship for some fully
hydrated unconsolidated natural and synthetic sands [4]. In
that study we adopted the convention that for similarly
shaped grains, (S/V)
P
is inversely proportional to the sand
grain diameter (d). In doing so, we were able to fit Eq. (1) to
R
1
values for different diameter sands, thus calculate for
the sands. The values used for the fit were proportional to
the electron spin resonance signal for the dry sands. This is a
reasonable find as the surface relaxivity is caused by par-
amagnetic impurities in the surface layer of atoms of the
grains. The concentration of paramagnetic impurities in the
entire grain volume, represented by the ESR signal, is pre-
sumed to be uniform throughout and the same as the concen-
tration in the outer surface layer governing .
A preliminary report indicated that this relationship may
not be true for all sands [10]. Surface weathering or surface
adsorption of paramagnetic metals may cause the concentra-

*Address correspondence to this author at the RIT Magnetic Resonance
laboratory, Center for Imaging Science, Rochester Institute of Technology,
54 Lomb Memorial Drive, Rochester, NY 14623-5604, USA; Tel:
585.475.2904; Fax: 585.475.5988; E-mail: jphsch@rit.edu
tion of these metals on the surface layer to differ from that
within the grain. The comparison made to exemplify this
difference is between an active beach sand from Asilomar,
CA USA, and two inland sands from Illinois USA. We pre-
sent the details of these findings in this paper.
In the previous paper, we reported R
1
values at proton
resonance frequencies () between 30 MHz and 10 kHz. This
data was used to extrapolate a value for R
1
and at = 2.5
kHz, the approximate resonance frequency of protons in the
magnetic field of the Earth. This extrapolation was risky
because the dispersion in R
1
with could either continue at
< 10 kHz or stop and R
1
level off. In the current presentation,
we have added a datum point at 1.9 kHz using an Earths
field NMR spectrometer that eliminated the need to extrapo-
late.
BACKGROUND
The R
1
of a nuclear spin system is influenced by time-
varying magnetic fields. In pure water, R
1
results from di-
pole-dipole interactions between water protons modulated by
the rotational motions. The frequency dependence of these
time varying magnetic fields is given by the spectral density
function, J(), for bulk water [11]. J() is flat over a broad
range of frequencies from zero to a frequency equal to the
inverse of the correlation time (
c
) for the rotational motion,
where J() decreases to zero. This change in J() is referred
to as a dispersion and plots of the R
1
value as a function of
are referred to as NMR dispersion plots. The R
1
of the
1
H
spins in pure water depends on the number of time-varying
magnetic fields at and 2 experienced by the nucleus [12].
Water molecules experiencing an electrical charge from ions
will possess a different
c
due to the electrostatic attraction
between the polar water molecule and the ion.
In the presence of paramagnetic ions, the water R
1
is also
influenced by electron-nuclear dipolar and contact interac-
tions between the nuclear spin of water hydrogens and the
electron spin of paramagnetic material [13,14]. The correla-
tion times for these interactions are influenced by a rota-
tional correlation time for a water molecule in the hydration
A Possible Difference in the Surface Relaxivity of Costal The Open Magnetic Resonance Journal, 2010, Volume 3 53
sphere of the paramagnetic material, the electron spin-lattice
relaxation time, the proton-electron hyperfine coupling con-
stant, and the electron spin-spin relaxation time.
The R
1
of water adsorbed on a surface is also influenced
by electron-nuclear dipolar and contact interactions, except
the interaction is between the nuclear spin of water and the
electron spin of paramagnetic material both in and on the
surface. These interactions are influenced by the correlation
times described previously for paramagnetic ions in solution,
plus ones for diffusion (
m
) and desorption (
s
) motions of
the water on and from the surface of a particle [15].
Randomly packed, unconsolidated, solid sand particles
form a network of pores connected by channels [16]. The
size of a pore is proportional to the diameter of the particles
[15]. When fully hydrated, water fills the pores and channels
between the particles. The mechanism of NMR spin relaxa-
tion in porous media is well established [1-3], and can be
divided into two limiting cases: fast-diffusion or surface-
limited, and slow-diffusion or diffusion-limited. In the fast-
diffusion case, the magnetization recovery for a single pore
is monoexponential and depends on the (S/V)
P
. In the slow-
diffusion regime, the dominant relaxation occurs at the sur-
face but the diffusion of spins to the surface is slow. In this
case for a single pore, the return of magnetization to equilib-
rium is multiexponential and depends on the pore shape.
McCall et al. [17] have presented modifications to the theory
for coupled-pore systems. Sands similar to the Asilomar
sand contain well-coupled pores of similar dimensions [4]
and hence we focus on the fast-diffusion case.
In the fast-diffusion case, R
1
is influenced by bulk water
and a thickness of water () adsorbed on the surface of the
pore, both with distinctly different R
1
values. On the time-
scale of the NMR relaxation measurement, water molecules
readily diffuse between the two environments. R
1
is a func-
tion of R
1
B
and the surface relaxation rate ( R
1
S
).
R
1
= R
1
B
+ R
1
S
S
V
P
(2)
Eqn. 2 is an alternative representation of eqn. 1 as the surface
relaxivity is defined as R
1
S
.
Similar to the relaxation in porous materials model of
eqns. 1 and 2, water in the presence of solvated diamagnetic
ions can be characterized as existing in two different envi-
ronments. There is structured water in the solvation shell of
the ions, and bulk water not experiencing the effect of the
ions. The fraction of structured water associated with the
salvation shells of the ions is , and the remaining fraction
(1-) is bulk water. On the NMR timescale, with rapid ex-
change between the two environments the measured relaxa-
tion rate for an aqueous solution of diamagnetic ions be-
comes
R
1
= 1 ( ) R
1
B
+R
1
H
(3)
where R
1
H
is the R
1
of the structured water in the hydration
shell [18,19]. R
1
H
is characterized by a unique
c
from di-
pole-dipole interactions between the water molecules in the
hydration shell.
In the fast-diffusion case, the J() from all the above in-
teractions contribute, although not equally, to the overall
frequency dependence of R
1
for hydrated sands. Fig. (1)
summarizes the frequency dependence of these interactions.
For bulk water systems at 20 C, the rotational
c
3.510
-12
s
[11], placing the dispersion from these motions at a
frequency well beyond the reach of NMR spectrometers. The
interaction of water protons with paramagnetic ions in solu-
tion or in the lattice can cause dispersions between approxi-
mately 10
5
and 10
11
s
-1
. The surface diffusion/desorption
interaction is unique in that it is characterized by a large con-
stant R
1
at < 1/
s
, a small constant R
1
at > 1/
m
, and a
logarithmic relation between 1/
s
and 1/
m
. The value of
m
is
~410
-10
s and
s
is less than ~110
-4
s [15]. The surface dif-
fusion/desorption interaction is also unique in that it pro-
duces a dispersion with a linear relationship between R
1
and
log() while the other interactions all produce power law
dispersions.

Fig. (1). Plots of the normalized spectral density function (J) as a function of
magnetic field reported as proton NMR frequency for a) proton-electron
hyperfine, b) surface diffusion/desorption, c) proton-electron dipolar, and d)
proton-proton dipolar interactions.

The surface relaxivity is caused by the electron-nuclear
dipolar and contact interactions between the
1
H nuclear spins
in water and paramagnetic materials on and in the sand
grains. These paramagnetic substances can be either ad-
sorbed on the surface [20], or randomly distributed through-
out the grain and hence exposed at the surface. With the lat-
ter, it is possible to assess the surface concentration using
ESR as the area of the ESR absorption signal is proportional
to the concentration of electron spins in the sample. For par-
amagnetic materials adsorbed on the surface, it is only possi-
ble to relate the ESR signal to when the concentration of
spins in the grains is known separately or zero.
Three natural quartz sands were compared: Asilomar
Beach, Ottawa, and Oregon Sands. The first is from the Asi-
lomar Beach in Asilomar, CA USA and these sand grains are
currently being formed from the erosion of the native bed-
rock. Both the Ottawa and Oregon sand grains were formed
0.001 0.1 10 1000 100000
H
B
o
(MHz)
J

a
b c
d
54 The Open Magnetic Resonance Journal, 2010, Volume 3 Hornak et al.
from the same erosive forces associated with a Paleozoic era
sea approximately 465 million years ago [21]. Since then,
these two sands have experienced varying degrees of surface
weathering.
The Asilomar Beach Sand was collected fully hydrated
with ocean water and used without sieving. This fully hy-
drated sand was used as is for the NMR studies. For ESR
and geometric measurements, the sand was rinsed with 18
Mcm deionized (DI) water and dried at 200 C. Ottawa
Sand (F110, US Silica, Berkeley Springs, WV, USA) and
Oregon Sand (7020 Granusil, Unimin Corp., New Canaan,
CT, USA) were acquired from the respective suppliers. The
Ottawa and Oregon sand samples were cleaned by rinsing
with DI water, oven dried at 200 C, and sieved using a me-
chanical 76 mm sieve shaker (SS-5, Gilson Co. Inc., Wor-
thington, OH) to obtain the diameters studied [4]. ESR and
geometric measurements were made on the dry samples,
while NMR was performed on the sands fully hydrated with
DI water.
The geometric properties of all the sand grains were
measured using an optical microscope (Eclipse E600PL,
Nikon) with image analysis software (analySIS, Olympus).
The average minimum and maximum diameters (d
min
and
d
max
) of a random sampling of 100 grains were measured and
used to calculate the aspect ratio (R
A
= d
max
/d
min
), the average
diameter, and uniformity (
d
/d) of the grains. All the geo-
metric properties are used to estimate similarities in (S/V)
P
.
The R
1
values of the Asilomar Sand were measured by
two techniques. R
1
values were measured at an Earths mag-
netic field of 44.6 T (1.9 kHz) using an Earths field NMR
spectrometer (Terranova-MRI, Magritek, Wellington, New
Zealand). This same instrument was used to measure the R
1

in its polarizing field of 18 mT (766 kHz). Both of these
measurements were made on 250 ml volume samples. R
1

values as a function of field strength were measured between
0.47 mT (20 kHz) and 0.19 T (8 MHz) using a field cycling
NMR spectrometer (SMARtracer, Stelar, Mede, Italy). Field
cycling measurements were made on samples in 10 mm
NMR tubes. The R
1
value for the Asilomar sand was interpo-
lated at 2.5 kHz from Fig. (2).

Fig. (2).
1
H NMR R1 values as a function of resonance frequency between
1.9 kHz and 9 MHz for fully hydrated Asilomar Beach sand, Asilomar
Beach ocean water, and deionized (DI) water as measured by an Earths
field (Magritek) and field cycling (Stelar) NMR spectrometers.
R
1
values for the Ottawa and Oregon Sands were taken
from [4]. This study measured R
1
as a function of d and .
Because of the high density of data points in d from this
study, our R
1
values for these two sands were calculated
from their fitted plots of R
1
vs. d at d = 340 m, and thus
assuring comparison values for similar diameter sand grains.
The paramagnetic metal content of the sand was deter-
mined using a low frequency electron spin resonance ESR
spectrometer [22] operating at 247 MHz with a magnetic
field sweep between 0 and 15 mT. Sand samples were pre-
pared by rinsing with 18 Mcm deionized water and dried.
Sand samples were placed in 2 cm diameter glass sample
tubes to a height that completely filled the ESR sample
probe. Conventional first-derivative ESR spectra were re-
corded. The relative number of paramagnetic spins per mass
of sand (N
m
) was calculated from the double integral of the
ESR spectrum (S) minus the same for an empty sample tube
(S
o
) all divided by the mass of sand filling the probe (m).
N
m
= (S - S
o
)/m (4)
Assuming similar density quartz sand grains, this N
m
is pro-
portional to the concentration of spins in a grain.
All three sands had similar aspect ratios and mean diame-
ters. (See Table 1). Based on this, the sand grains should
pack similarly and have similar (S/V)
P
ratios. However, the
distribution of grain diameters in the Asilomar Sand was
greater than that for the Ottawa and Oregon Sand samples,
because the Asilomar Sand was not sieved. This 2.3 times
larger distribution of d will cause a difference in the packing
and (S/V)
P
, which we will address later.
Table 1. Comparison of Hydrated Sands at = 2.5 kHz
Property Asilomar Ottawa
a
Oregon
a

d (m) 340 340 340
d/d 0.30 0.13 0.13
RA 1.5 1.5 1.4
Relative ESR Signal 0.9 1.0 1.7
Relative 2.5 1.0 1.6
(R1-R1B) (s
-1
) 0.80 0.32 0.49
a
Data at specified diameter were interpolated from [4].

The R
1
values for Asilomar Beach sand hydrated with
ocean water, Asilomar Beach ocean water, and DI water as a
function of proton resonance frequency are presented in Fig.
(2). Both Stelar and Magritek instruments produced consis-
tent data at their overlapping frequency of 766 kHz. Com-
pared to 18 Mcm deionized water, the ocean water shows
a higher R
1
at all values. This is attributable the 0.5 M
diamagnetic salt ions in sea water acting through eqn. 3, and
not the < 30 nM paramagnetic ions [23]. The slight slope of
the line for the two bulk water samples is not predicted by
theory and may reflect measurement uncertainty.
The R
1
of the hydrated sand grains was greater than that
for ocean water and increased with decreasing resonance

Asilomar Beach Data
0.1
1
10
0.001 0.01 0.1 1 10
(MHz)
R
1

(
s
-
1
)
Sand (Stelar)
Sand (Magritek)
Ocean Water (Stelar)
Ocean Water (Magritek)
DI Water (Stelar)
DI Water (Magritek)
A Possible Difference in the Surface Relaxivity of Costal The Open Magnetic Resonance Journal, 2010, Volume 3 55
frequency to about 20 kHz. The dispersion in R
1
between 20
kHz and 8 MHz is the result of surface relaxation due to in-
teractions of the water with paramagnetic impurities in the
sand grains and is consistent with theory [15]. It was not
possible to determine if the data followed a power law or
log() relationship as both forms fit the data equally well.
Below 20 kHz, R
1
appears to level off. The leveling off is
significant because it sets a lower limit on the dispersion.
Extrapolation of the dispersion to the resonance frequency of
1
H in the Earths magnetic field may yield a higher R
1
. It
may be more appropriate to use the R
1
value at lowest fre-
quency of the field cycling studies, i.e. 10 or 20 kHz, as the
extrapolated R
1
at 2.5 kHz then to extend the dispersion be-
yond the 10 kHz R
1
value. This hints that the extrapolation in
the previous study may have produced slightly higher values
at 2.5 kHz then actual values.
The R
1
value of the Asilomar Sand was compared to that
of the previously reported Ottawa and Oregon, IL Sands [4].
Fig. (3) shows a plot of (R
1
R
1B
) vs. log(d) for the Ottawa
and Oregon Sands. The data was fit with Eq. (1) to obtain the
relative values of 1.0 and 1.6 respectively. (See Table 1).
These values were proportional to the relative ESR signals
of 1.0 and 1.7. The R
1
values were measured for several dif-
ferent diameter samples; however, these were not the same
as the Asilomar Sand grain diameter. Therefore, the (R
1
R
1B
)
values of Table 1 are based on the fit to the data in Fig. (3) at
d = 340 m. For comparison purposes, the datum point for
the Asilomar Sand in Fig. (3) is displayed with a line seg-
ment for a = 2.5. This relative value was determined by
fitting eqn. 1 to the point.

Fig. (3).
1
H NMR (R1R1B) values as a function of diameter (d) for fully
hydrated Oregon (), Ottawa (), and Asilomar Beach () Sands. Solid
lines are best fits of the data using Eq. (1). The line segment through the
Asilomar Sand point is provided as a reference for the value.

The previous study of hydrated Ottawa and Oregon sands
showed that the measured (R
1
-R
1B
) values correlated well
with the ESR signal and could thus be used to predict [4].
In our study, the (R
1
-R
1B
) value of hydrated Asilomar Sand is
greater than that of inland Ottawa and Oregon Sands. (See
Table 1). As per Eq. (1), the difference must be due to (S/V)
P

or . The similar d and R
A
values would assure very similar
packing of the grains if the
d
/d values were similar. The
literature suggests that the difference in d must be greater
than 6.5 to see a significant change in the porosity [24,25].
Therefore, the 2.3 times larger
d
may not cause a significant
change in (S/V)
P
and is not the primary cause of the larger
(R
1
-R
1B
) for Asilomar Sand. This points to as the cause of
the larger (R
1
-R
1B
) for the Asilomar Sand.
The ESR signal of the Ottawa and Oregon Sands corre-
late well with the value needed to fit the data of Fig. (3),
but the Asilomar Sand value does not. This observation
leads us to believe that the concentration of paramagnetic
material on the surface of the grains of sand may be different
from the concentration in the center. The ESR signal is pro-
portional to the total paramagnetic concentration. Either a
higher surface concentration of paramagnetic material in the
Asilomar Sand, or a lower surface concentration of par-
amagnetic material in the Ottawa and Oregon Sands could
account for the difference. The former could be attributed to
paramagnetic material from the seawater adhering to the
surface of the grains. The rinsing of the sand for ESR meas-
urements and the ESR averaging of the signal from the sur-
face and interior of the grains would account for the higher
than predicted by ESR. The latter cause could come about
from repeated weathering of the surface of the inland sand
grains. Acid washing the surface of the Asilomar Sands
might remove the adsorbed and surface layer paramagnetic
materials, however rehydrating with ocean water might
cause some ions to be readsorbed. The best way to distin-
guish between the two causes is to use an analytical tech-
nique that measures only the surface concentration of these
trace paramagnetic metals.
CONCLUSIONS
Unlike in a previous study where the ESR signal was
found to be proportional to the surface relaxivity of several
sands and glass spheres, in this study the ESR signal was
found not to be proportional to the surface relaxivity of a
specific ocean beach sand hydrated with ocean water. Al-
though (S/V)
P
could not be completely ruled out as the cause,
it is believed that a difference between the surface and over-
all concentration of paramagnetic material for the sand
grains is most probably the cause. The difference may be due
to paramagnetic ions found in sea water being adsorbed on
the surface of the grains of Asilomar Sand, or the weathering
of paramagnetic ions out of the surface layer in the geologi-
cally older inland sands. More studies are needed to confirm
the cause and determine the best analytical technique for
measuring the surface relaxivity.
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0.1
1
10
100 1000
d (m)
(
R
1
-
R
1
B
)

(
s
-
1
)
Oregon, IL Sand
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Asilomar Beach, CA

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Received: July 28, 2009 Revised: October 30, 2009 Accepted: November 04, 2009

Hornak et al.; Licensee Bentham Open.



1874-7698/10 2010 Bentham Open
Open Access
Relaxation in a Natural Soil: Comparison of Relaxometric Imaging, T
1

T
2
Correlation and Fast-Field Cycling NMR
S. Haber-Pohlmeier*
,1
, S. Stapf
2
, D. van Dusschoten
3
and A. Pohlmeier
4

1
ITMC, RWTH Aachen University, Worringer Weg 1, Aachen, Germany;
2
Institute of Physics, Technical University
Ilmenau, Germany;
3
ICG3, Research Centre Jlich, German;
4
ICG4, Research Centre Jlich, Germany
Abstract: Longitudinal and transverse relaxation times are used to characterise the pore system of a natural Kalden-
kirchen sandy loam. Here we present new results obtained by relaxometric imaging (MEMS) and two-dimensional T
1
-T
2

correlation relaxometry, and compare these with available T
1
- relaxation time distributions of water obtained by the analy-
sis of fast field cycling relaxometry (FFC) data. The soil shows relatively broad bimodal distribution functions P(T
1
) and
P(T
2
) with a T
1
/T
2
ratio of about 2:1. The average T
1
as well as the spatial distribution, which are obtained from the re-
laxometric imaging corresponds well to the relaxometric results. From the analysis of the field dependent FFC data at low
field including T
1
data obtained at high field the basic locally averaged relaxation mechanism is derived from the disper-
sion curve, i.e. the dependence of the relaxation rate from the magnetic field strength over five orders of magnitudes.
From this we conclude that two-dimensional diffusion at locally flat surfaces controls the relaxation, i.e. the shapes of the
distribution functions are controlled by surface relaxation.
Keywords: NMR, relaxometry, imaging, pore size distribution, Brownstein Tarr, T
1
-T
2
correlation.
INTRODUCTION
Soils are natural porous media of highest importance for
food production and maintenance of water resources. For
these functions a prominent property is their ability to retain
and transport water which is mainly controlled by pore size
distribution. The latter is related to NMR relaxation times of
the water molecules filling the pores, for which different
measurement methods can be used. Most widespread are
Carr Purcell Meiboom Gill (CPMG) for transverse relaxation
and inversion or saturation recovery (IR, SR) for longitudi-
nal relaxation (T
1
) [1]. Besides this, T
1
can also be deter-
mined by Fast Field Cycling (FFC) [2] which allows investi-
gations at variable field strength. Relaxometric methods can
also be combined among themselves yielding correlation
maps [3] or with imaging (MRI) leading to spatially resolved
T
1
and T
2
times, which can also be analyzed statistically [4-
9]. Generally, imaging methods contain more information
than relaxation methods, since imaging resolves the informa-
tion in two or three dimensions which is especially important
for heterogeneous samples such as natural soil. But this is
often paid by increased noise and a much smaller number of
data points.
The aim of this study is to investigate the multimodal na-
ture of the relaxation distribution functions observed in re-
laxation experiments of macroscopic samples at low fields
and to find an answer to the question whether or not they are
caused by macroscopic structures like fractures or by the
inherent microscopic heterogeneity. For this purpose we
measure T
1
and T
2
maps by relaxometric imaging and two-
dimensional T
1
-T
2
correlation maps and compare them with

*Address correspondence to this author at the ITMC, RWTH Aachen Uni-
versity, Worringer Weg 1, Aachen, Germany; Tel: +49(241) 80-264247;
Fax: +49(241) 80-22185; E-mail: s.haber-pohlmeier@fz-juelich.de
available data from fast field cycling relaxometry (FFC) ex-
periments of a saturated natural sandy loam from Kalden-
kirchen [10]. This will help in the future to better assess the
information obtainable from magnetic resonance imaging
which aims for understanding flow processes in natural soils.
MATERIAL AND METHODS
Sample Preparation
The Kaldenkirchen soil sample (73% sand, 23% silt, 4%
clay, 0.25% Fe, 0.02% Mn, , 1% TOC) was sieved (<2mm)
and filled, air-dry, into 9x100mm
2
quartz glass cuvettes
which are closed at the bottom by a porous glass filter plate.
The cuvettes were then centrifuged for 30 min at 3000rpm
and finally wetted from the bottom for 48 h. The maximum
water content was = 0.45. Then the sample was sealed and
measured by fast field cycling NMR relaxometry (FFC)[10],
IR-CPMG, and imaged by a multislice-multiecho method
(IR-MEMS).
Fast Field Cycling Relaxometry (FFC)
This method investigates the relaxation of an excited spin
ensemble along the direction of the main magnetic field after
a field jump [2]. The timing diagram is shown in Fig. (1). At
the beginning the system is in thermal equilibrium corre-
sponding to B=B
pol
or B=0, respectively. Then the field
strength is switched quickly to B
rlx
, but the spin ensemble
does not follow instantaneously, i.e. the restoration of the
new thermal equilibrium obeys a first order kinetic law with
a characteristic relaxation time T
1
. The polarization state of
the ensemble is probed after varying time intervals , during
which the sample is left at the magnetic field strength B
relax
.
This probing is performed by switching the magnetic field
strength to B
acq
and subsequently applying a 90 radio-
frequency pulse. The free induction decay (FID) following
this pulse is recorded and analyzed off-line in order to obtain
58 The Open Magnetic Resonance Journal, 2010, Volume 3 Haber-Pohlmeier et al.
its amplitude, which is proportional to the magnetisation
M
z
(). A complete relaxation curve is then obtained by
repeating the measurement cycle for different values of . In
ideally homogeneous systems M
z
() is a single exponential
function with a unique relaxation time T
1
, in heterogeneous
porous media multiple exponential functions are expected.
The whole procedure may be repeated for different values of
B
relax
. All FFC measurements were performed on a Stelar
Spinmaster (Stelar, Mede, Italy). T
1
relaxation curves were
monitored at relaxation fields corresponding to Larmor fre-
quencies of = B
rlx
/2 = 20, 10.6, 5.6, 3, 1.6, 0.823, 0.435,
0.23, 0.121, 0.064, 0.034, 0.018, 0.009, and 0.005 MHz ( is
the gyromagnetic ratio of
1
H). The data are analysed by 1D
inverse Laplace transformation [3].

Fig. (1). Fast Field Cycling Method.

IR-MEMS NMR Imaging
The imaging experiments were performed on a 7T verti-
cal bore magnet (Oxford Instruments, Oxford, UK),
equipped with a Bruker (Karlsruhe, Germany) mini-imaging
gradient set (G
max
=300 mT/m), a 38mm rf birdcage coil, and
a Maran DRX console. The used imaging sequence was a
inversion recovery multiecho multislice sequence (IR-
MEMS), which allows spatially resolved simultaneous de-
termination of T
1
and T
2
relaxation times. A simplified tim-
ing diagram is displayed in Fig. (2).

Fig. (2). Simplified IR-MEMS pulse sequence.
The basic idea of MEMS is a preparatory initial 180
pulse which inverts all magnetisation into z direction. After
a period t
In
during which longitudinal relaxation evolves, a
90 slice selective soft pulse is applied which is followed by
a CPMG echo train for the T
2
determination. This is repeated
for several different inversion time periods. Due to the addi-
tional switching of phase ph and read-out ro encoding
gradients the complete inversion-recovery CPMG pulse se-
quence becomes spatially resolved [3].
The signal intensity in each voxel obeys the following
relation:

S(x, y, z, T
In
, T
E
) = S
0
(x, y, z) 1 F exp(
t
In
T
1
exp(
nt
E
T
2, app
(1)
with the inversion time t
In
, the echo time t
E
, the number of
echoes n, and the longitudinal and apparent transverse re-
laxation times, T
1
, and T
2,app
. The transverse relaxation time
T
2
is denoted as apparent since it comprises the true T
2
plus
surface and diffusional effects. The prefactor F is theoretic-
cally 2, but in practice it should be fitted to the data in order
to account for fast relaxation processes during the dead time
of the receiver and effects of imperfect inversion pulses.
Since soils possess quite fast apparent transverse relaxation
times, T
2,app
, we used the shortest adjustable value of t
E
= 1.5
ms, which is paid by a relatively high slice thickness of
3mm. Further parameters were 0.009s < t
In
< 1 s, t
R
= 1.8s,
FOV = 64x32 (32 x 16mm), and 14 axial slices scanned in
interleaved mode. Original data S(x,y,z,t
In
,nt
E
) obtained for
each voxel were fitted by Eq. 2, yielding maps (2D represen-
tations for chosen slices) of the physical quantities S
0
, T
1
,
and T
2,app
.
T
1
-T
2
Correlation
The relation between longitudinal and transverse relaxa-
tion times can also be combined in a T
1
-T
2
single NMR re-
laxation experiment. This is the non-spatially resolved ver-
sion of IR-MEMS imaging and for the data analysis also Eq.
2 is valid. We have applied this method on the Kalden-
kirchen soil sample described above in a 24 MHz Halbach
low field scanner operated by a Stelar PC-NMR console
(Stelar, Mede, Italy). t
E
= 0.3ms, n = 512, 0.001s < t
In
< 1s.
The data are analyzed by 2D inverse Laplace transformation
using Eq. (1) as kernel for Eq. (2):

M
z
(t ) =
[
P(T
1
,T
2
) 1 2exp(
t
In
T
1
_
,
[

exp(
nt
E
T
2
_
,
dT
1
dT
2
+ err
(2)
P(T
1
, T
2
) is the unknown distribution function, the terms
in brackets constitute the kernel, t
In
is the inversion time, t
E
is
the echo time, n is the number of echoes, and err is an un-
known error term [3, 11].
RESULTS
Fig. (3) shows T
1
and T
2
maps of four chosen axial slices
of the saturated Kaldenkirchen soil sample. All regions from
the soil give measurable and analyzable NMR signals. This
means that this type of soil (sandy loam) is investigable by
MRI which is not trivial since many soils show much shorter
B
pol

B
rlx

B
acq

P
90

FID
Transm./Acquis.

var.
var. B
rlx
rf
n
Echo

ro
sl
ph
n
Ti

phase-steps
T
In
T
E

Relaxation in a Natural Soil The Open Magnetic Resonance Journal, 2010, Volume 3 59
transverse relaxation times due to small pore sizes and high
content of paramagnetic ions [12]. The soil shows T
1
in the
range of about 50 ms and T
2
of about 3 ms. Some spots with
faster (green) and slower T
1
relaxation times (violet-white)
are also present in the sample, especially near the top. T
2

appears more homogeneously. The long T
1
values in the top
layer are caused by the somewhat smaller local packing den-
sity corresponding to larger pores.

Fig. (3). T
1
and T
2
maps for the Kaldenkirchen soil sample at 7 T
(
H
= 300 MHz).

From the T
1
and T
2
maps relaxation time distributions are
extracted by calculation of histograms of the populations of
relaxation times in the soil, excluding the porous plate layer
(Fig. 4). T
2
shows a comparably narrow distribution in the
range between 1 and 8 ms, with an average of 2 ms. But also
voxels with higher T
2
(10 to 20 ms) exist, which are located
near the edges. This is most probably due to packing faults at
the soil-wall interface, where pores are larger. Also T
1
in
these regions is longer. These effects may be interpreted by
the Brownstein-Tarr model [13], which relates 1/T
1,2
to the
inverse pore size parameter Surface/Volume S/V, see Eq. 3
[10].

Fig. (4). T
1
(red) and T
2
(blue) histograms of the maps in Fig. (3).
T
1
shows a bimodal distribution, with a sharp peak at 7
ms and a broad mode between 10 and 300 ms. The sharp
peak is near the shortest inversion time, and therefore must
be interpreted with caution. It comprises all longitudinal re-
laxation processes faster than 10 ms, which are present, but
not further resolved. The main mode is approximately log-
normal distributed with T
1,mean
= 80 ms and a half width of
log (T
1
) = 0.3. T
2
depends strongly on t
E
, so this is hard to
compare. However, the reported relaxation times are in the
range reported by Hall et al. [12] for several soils, e.g.
Korkusova Hut sandy loam which has comparable texture
with T
2
= 0.9 ms for t
E
=1ms and t
1
= 90 ms at 40% satura-
tion. It should be noted that Votrubova et al. [5] found for
saturated soil also by statistical analysis of T
1
maps an aver-
age T
1
of 600 ms with a broad distribution over one order of
magnitude. Issa et al. calculated T
1
relaxation time distribu-
tions for rocks in the same range [4]. Also a forest soil [14]
with comparable texture shows similar relaxation times (5
and 45 ms at t
E
= 0.3ms). The T
1
relaxation has been further
investigated at lower Larmor frequencies between 5 kHz and
20 MHz by the FFC method.
Fig. (5) shows the measured relaxation curves, which are
further analyzed by inverse Laplace transformation. The re-
sults are presented in Fig. (6). Clearly distinguishable are
three processes: A broad main mode with average T
1
of 70
ms, which is accompanied by a faster relaxing component at
about 10ms. Additionally, a small but very fast component at
2 ms is observed. Note that the limit of the shortest detect-
able T
1
component in the FFC experiments is about 1 ms. All
modes shift simultaneously to faster times with decreasing
Larmor frequency.

Fig. (5). T
1
relaxation curves at different Larmor frequencies for the
saturated Kaldenkirchen soil sample, (modified, from [10]).

The data have already been analyzed further in terms of
pore size distribution [10], where we found that the distribu-
tion of T
1
between 10 and 200 ms corresponds to pore sizes
between one and some tens of microns using the Brown-
stein-Tarr model, see below. Here we include results from
the high-field measurement into the analysis. The mean re-
laxation times for the main mode from FFC and MEMS
measurements are plotted in Fig. (7). All data including the
result at 300 MHz lie on one line. This indicates that the dis-

7 mm

0 s 0.1 0.2 0.3 0ms 10 20
a) T
1
b) T
2

32 mm
1 10 100 1000
0
100
200
0
50
100

P
o
p
u
l
a
t
i
o
n

T
2
T
1,2
/ ms

P
o
p
u
l
a
t
i
o
n

T
1
0 100 200 300
0
2
4
6
8

N
M
R

s
i
g
n
a
l

x
1
0
4

(
a
.
u
.
)
(ms)
= 20 MHz
= 5 kHz
persive behaviour follows the same frequency dependence in
the whole observed range. The comparably weak dispersion
and the linear relation between 1/T
1
and the logarithm of the
Larmor frequency
H
indicates that the surface relaxation
mechanism is controlled by local 2D diffusion processes at
the pore walls [15].

Fig. (7). T
1
relaxation at different Larmor frequencies from FFC
(Fig. 6) and MEMS T
1
map (Fig. (4)) (modified, from [10]).

Finally, the results are compared to a T
1
-T
2
correlation
experiment at
H
= 24.3 MHz which is the non-spatially re-
solved version of the IR-MEMS experiment. The result is
shown in Fig. (8). On the right facing axis labelled nt
E

CPMG echoes are plotted, and the second, left facing axis,
labelled t
In
, contains the different t
In
values. The diagram
should be read as an array of CPMG curves as a function of
increasing inversion times t
In
, during which T
1
relaxation
evolves. The data are further analyzed by 2D inverse Laplace
transformation using the program of Y.-Q. Song [3]. Fig. (9)
shows bimodal distributions for both T
1
and T
2
which indi-
cates two distinct pore size classes. The low amplitude spots
at the limits of the inversion range are due to noise in the
experimental data. The maxima for T
1
are at 20 and 90 ms
and they correspond well to the FFC data for 20 MHz and
the IR-MEMS data at 300 MHz (Fig. (7)). The main trans-
verse relaxation modes at 9 and 50 ms are much slower than
the T
2
modes identified at 300 MHz. As the echo time is
much shorter at low field and the internal gradients are
higher at high field this may be explained by the effect of
molecular diffusion at high field.

Fig. (8). T
1
-T
2
correlation experiment on saturated Kaldenkirchen
soil at
H
= 24.3 MHz.

Fig. (9). T
1
-T
2
correlation diagram of the data in Fig. (8) obtained
by inverse 2D Laplace transformation, Eq. 2.

With decreasing T
1
(corresponding to decreasing pore
sizes) the 2D distribution function shows an increasing ratio
T
1
/T
2
from two to three. These ratios agree well with several
data obtained for porous media like rocks [16] and cements
[17] and with theoretical considerations [17, 18]. An impor-
tant observation is the lack of any detectable cross-peak con-
tribution. A cross peak would indicate that for a give value

Fig. (6). T
1
relaxation spectra for Kaldenkirchen soil at different
Larmor frequencies, normalized and offsetted (modified, from
[10]).
1 1 0 1 0 0 1 0 0 0
r l x
= 5 k H z
r lx
= 2 0 M H z

D
(
T
1
)
,

a
.
u
.
,

s
h
i
f
t
e
d
T
1
/ m s
0.01 0.1 1 10 100
0.00
0.01
0.02
0.03
0.10
0.15
0.20

T
1
-1
(FFC) long
T
1
-1
(FFC) short
T
1
-1
, av.IR-MEMS
T
1
-
1

/
m
s
-
1
/ MHz
nT
E
(s)
T
In
(s)

Relaxation in a Natural Soil The Open Magnetic Resonance Journal, 2010, Volume 3 61
of T
1
two or more values of T
2
exist indicating multiple
transverse relaxation mechanisms, so that T
2
is affected also
by parameters other than pore size.
The general way to transform T
1
and T
2
distribution func-
tions into pore sizes is the scaling by means of the Brown-
stein-Tarr equation:
V
S
T T
bulk
2 , 1
, 2 , 1 2 , 1
1 1
+ =
, (3)
incorporating an average surface relaxivity parameter
1,
2
, which on its side is obtained from the average T
1
, or T
2
.
This is obtained from the average surface/volume ratio S/V =
8300 cm
-1
(from BET measurements, see [10]) as
1
=
0.0023 cm s
-1
.
2
depends on t
E
, we obtained
2
= 0.0063 cm
s
-1
for the conditions of this investigation. Using these data
the results from the 2D experiment in Fig. (9) can be recalcu-
lated into pore size distributions, shown in Fig. (10). The
curves agree very well, although that obtained from T
2
is
somehow broader.

Fig. (10). Pore diameter distributions obtained by projections on the
T
1
and T
2
axis from the 2D experiment in Fig. (9). The relaxation
times were rescaled to pore size distributions by means of the
Brownstein-Tarr equation, the average S/V ratio from BET meas-
urements [5], and assuming the capillary tube model.

The T
2
distribution obtained from imaging experiments is
restricted due to t
E
. A larger fraction of water in small pores
relaxes during the first t
E
period; a rescaling of T
2
distribu-
tion is meaningless, since it is strongly affected by internal
gradients. One only can estimate the pore size limit, below
which water is not detectable by MRI with these settings.
With T
2,av
= 2 ms, one obtains
2,300 MHz
= 0.06 cm s
-1
.
Choosing T
2
= t
E
for the limiting value, one obtains d
limit
=
4V/S = 4
2
/(1/T
2,limit
-1/T
2,bulk
) 4 m, assuming cylindrical
pores. Please note also that this pore size limit corresponds
to about the fast mode. A similar estimation for T
1
yields
1,300 MHz
= 0.0017 cm s
-1
corresponding to a lower pore size
limit of about 0.5 m, if one uses a fastest detectable longi-
tudinal relaxation time of 0.008s.
CONCLUSIONS
Kaldenkirchen sandy loam shows broad bimodal distri-
bution functions of the longitudinal relaxation times T
1
over
a large range of Larmor frequencies. A further very fast
mode at around 2 ms is present but not well resolved. This
short component probably corresponds to the clay compo-
nents and represents strongly confined water in this phase.
Similar short values have already been found in a different
clayey soil [10]. The slow modes of T
1
at low magnetic
fields correspond to the slow mode observed spatially re-
solved at high field (300 MHz). These modes are due to the
relaxation in pores with a size in the range between 1 to
some tens microns. The two slow modes (10 and 80 ms) are
logarithmically dependent on the Larmor-frequency in the
low field range and this continues for the slowest mode at
high field of 300 MHz. According to Korb [15] this linear
dependence on the logarithm of the Larmor frequency is
interpreted as locally two-dimensional diffusion, where the
basic relaxation mechanism is dipole-dipole interaction of
water molecules with paramagnetic centres at the pore walls.
The spatially resolved relaxometric imaging method IR-
MEMS generally yields additional information to the re-
laxometric measurements, although with inferior signal/noise
ratio. This is mainly due to the smaller volumes over which
the signal is integrated, i.e. a single voxel rather than the
whole sample. The validity of the data is proven by the
agreement between the average values from T
1
maps with T
1

determined relaxometrically. But T
1
-mapping reveals struc-
tures which possess locally higher T
1
values in regions near
the walls as well as near the top surface, i.e. the local pores
are larger. This is due to packing faults near the walls and
near the surface which are unavoidable for such natural sam-
ples.
The texture of the soil is finer than 2mm, no large grains
are present, but aggregates of course. Water may penetrate
into these aggregates, and contribute to the relaxation. The
usage of 9 mm cuvettes and repacking the soil will definitely
change the size of interaggregate pores. However, such pores
are mainly macropores. So the macroporous structure of the
soil will be changed due to the preparation and sample size.
The meso- and microporous structures will be affected much
less, so the relaxation time and pore size distributions reflect
mainly this meso- and microporous structure, which are most
responsible for the water storage of the soil, since the
macropores are emptied firstly.
This study shows that T
1
mapping is a valuable tool for
upcoming investigations of local pore size distributions in
natural, i.e. undisturbed, soil cores for e.g. detection of pref-
erential flow paths.
ACKNOWLEDGEMENTS
We thank N. Hermes, ICG-4, Research Centre Jlich for
technical assistance and the DFG (SFB Transregional Col-
laborative Research Centre 32, PO-746-2/1 and Sta 511-4/1)
for financial support.
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nance, petrophysical and logging applications. Pergamon:
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Nucl Magn Res Spectrosc 2004; 44: 257-320.
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Straley C. T1-T2 correlation spectra obtained using a fast two-
dimensional laplace inversion. J Magn Reson 2002; 154: 261-8.
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ponded infiltration into structured soil: a magnetic resonance imag-
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[6] Edzes HT, van Dusschoten D, Van As H. Quantitative T-2 imaging
of plant tissues by means of multi-echo MRI microscopy. Magn
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[7] Belliveau SM, Henselwood TL, Langford CH. Soil wetting proc-
esses studied by magnetic resonance imaging: correlated study of
contaminant uptake. Environ Sci Technol 2000; 34: 2439-45.
[8] Cislerova M, Votrubova J, Vogel T, Amin MHG, Hall LD. Mag-
netic resonance imaging and preferential flow in soils. In: Van
Genuchten MT, Leij FJ, Wu L, Eds. Characterization and meas-
urement of the hydraulic properties of unsaturated porous media;
U.S. Salinity Laboratory: Riverside CA 1997; pp. 397-411.
[9] Davies S, Hartwick H, Roberts D, Spowich K, Packer KJ. Quanti-
fication of oil and water in preserved rock by NMR spectroscopy
and imaging. Magn Reson Imaging 1994; 12: 349-53.
NMR relaxometry study of natural soils. Vadose Zone J 2009; 8:
735-42.
[11] Provencher SW. A constrained regularization method for inverting
data represented by linear algebraic or integral-equations. Comput
Phys Commun 1982; 27: 213-27.
[12] Hall LD, Amin MHG, Dougherty E, et al. MR properties of water
in saturated soils and resulting loss of MRI signal in water content
detection at 2 tesla. Geoderma 1997; 80: 431-48.
[13] Barrie PJ. Characterization of porous media using NMR methods.
Ann Rep NMR Spectrosc 2000; 41: 265-316.
[14] Schaumann GE, Hobley E, Hurrass J, Rotard W. H-NMR re-
laxometry to monitor wetting and swelling kinetics in high-organic
matter soils. Plant Soil 2005; 275: 1-20.
[15] Korb JP, Bryant R. Magnetic relaxation dispersion in porous and
dynamically heterogeneous materials. Adv Inorg Chem 2005; 57:
293-326.
[16] Schoenfelder W, Glaeser HR, Mitreiter I, Stallmach F. Two-
dimensional NMR relaxometry study of pore space characteristics
of carbonate rocks from a Permian aquifer. J Appl Geophys 2008;
65: 21-9.
[17] McDonald PJ, Korb JP, Mitchell J, Monteilhet L. Surface relaxa-
tion an chemical exchange in hydrating cement pastes: a two di-
mensional NMR relaxation study. Phys Rev E Stat Nonlin Soft
Matter Phys 2005; 72: 011409.
[18] Kleinberg RL, Farooqui SA, Horsefield MA. T1/T2 ration and
frequency dependence of NMR relaxation in porous sedimentary
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Received: August 17, 2009 Revised: December 11, 2009 Accepted: December 11, 2009

Haber-Pohlmeier et al.; Licensee Bentham Open.


1874-7698/10 2010 Bentham Open
Open Access
Low-Field NMR of Water in Model Soils
Oscar Sucre*
,1
, Federico Casanova
1
, Andreas Pohlmeier
2
and Bernhard Bluemich
1

1
ITMC RWTH-Aachen University, Aachen, D-52056, Germany
2
Forschungszentrum Jlich, Jlich, D-52425, Germany
Abstract: The evolution of water contained in soils is a physical phenomenon of importance in soil science and climatol-
ogy. This work presents preliminary results from the use of mobile NMR to measure moisture in soil columns. To demon-
strate the ability of the NMR technique to follow the drainage process of water in model soils, moisture measurements
were performed at a certain depth with a mobile NMR sensor during an one-step outflow experiment. The NMR sensor
exhibits a cylindrical geometry incorporating the principle of the u-shaped NMR-MOUSE. It could be raised and lowered
inside a plastic tube in the soil column similar to a wire-line logging tool. Working with a frequency of 8.8 MHz, the sen-
sitive volume lies 3 mm deep into the soil and 2 mm away from the tube walls, making the technique truly non-invasive.
For the purpose of quantitative analysis, the temporal evolution of moisture (described by the Richards Equation) was
modelled with the Hydrus1D Program. Out of these simulations, an assessment of the hydraulic parameters (Ks, , n of
the Van Genuchten model) of the soil was achieved.
Keywords: Low field NMR, moisture sensor, model soils, drainage, richards equation.
1. INTRODUCTION
The question of how water moves in soils is of impor-
tance in agriculture and climatological sciences.
Being a partially saturated porous medium, soil in the
vadose zone can either stop or permit the transport of water
depending on gravity, surface tension and evaporation. Prob-
ing of moisture on field plays a central role in addressing
these issues, and several techniques have gained acceptance
in the soil science community to perform that measurement:
Time Domain Reflectometry (TDR) [1] and Neutron Absor-
tion [2] among others.
The application of Nuclear Magnetic Resonance (NMR)
in soils is not new, as it has been applied to gain information
about the pore size distribution [3, 4], in the search of aqui-
fers [5] or to study the chemistry of soil organic matter [6,
7]. However as far as we know, little has been done to de-
velop an NMR instrument that can be deployed on field to
measure local values of soil moisture, taking advantage of
the non-destructive nature this technique possesses. In this
sense, NMR at low fields has just begun to make its way
among the above mentioned techniques. It can even show
some comparative advantages over TDR: As the signal to
probe water comes from the atomic nuclei (in case of water,
simply protons), it is not affected by any ions in solution, so
the NMR technique happens to be immune to any salt con-
tent in water absorbed in soil. Furthermore, due to the reso-
nance condition and the inhomogeneous magnetic fields
generated the sensor senses water in a very well defined vol-
ume [8], making the measure well resolved spatially. For
those reasons we believe the NMR can become standard to

*Address correspondence to this author at the ITMC RWTH-Aachen
University, Aachen, D-52056, Germany; Tel: 00-49-241-8026423;
E-mail: osucre@mc.rwth-aachen.de
sense moisture among soil scientist. It has even reached a
mature stage in its development as a logging tool for the oil
industry [9, 10]. Undeniably NMR is no excepted from ex-
perimental diffculties: low inherent signal-to-noise ratio con-
stitutes its main drawback. Common way to improve this
ratio is to average over repeatedly acquired data, however
that can make the whole profiling of soil moisture costly in
terms of time.
This article reports progress in probing soil moisture by
low field NMR. The work was performed as a part of the
interdisciplinary project Transregio 32 [11] (funded by the
German Research Council DFG): the drainage processes in a
first column filled with FH31 model soil and in a second
column with W3 model soils under known boundary condi-
tions were investigated with a newly developed low-field
NMR sensor: the slim-line NMR logging tool. The moisture
profile before and after drainage, and the moisture loss at
certain depth during drainage were thus recorded with this
instrument. To validate the results, the associated drainage
problem is solved numerically, producing a theoretical curve
that can be compared to the experimental results. By chang-
ing soil hydraulical parameters in the numerical solution the
better correspondence between theory and experiment is
found and the so obtained values are compared to reported in
the literature.
2. EXPERIMENT AND MATERIAL
The experimental setup is shown in Fig. (1) and consists
of two concentrical tubes, being the space between them
filled with the model soil to research. This arrangement or
soil "column" stays in a water tank. A copper fabric was
wound around the outer tube to shield it from electromag-
netic noise. The central inner column has a diameter of 31
mm and walls 1 mm thick. The NMR sensor moves within
the inner tube. Initially the model soil is completely saturated
by raising the water level in the tank up to top of the column.
64 The Open Magnetic Resonance Journal, 2010, Volume 3 Sucre et al.
The top of the column is then closed with a rubber seal that
allows the entrance of air (necessary for pressure balance
during drainage) and limits evaporation as well. The bottom
plate is connected to the water tank through perforations 4
mm wide and covered by a sheet of filter paper. This lets the
water drain out from the column and gives hold to model soil
lying thereupon. To obtain a complete profile of the satura-
tion along the column the sensor is displaced with a stepper
motor from the bottom to the top while NMR signal is ac-
quired.

Fig. (1). Setup for soil drainage experiments.

Two experiments of drainage were performed, one on
coarse sand FH31 (distribution of grain size classes 2%
(>0.72mm), 8% (0.71-0.5mm), 30% (0.5-0.355mm), 41%
(0.36-0.25mm), 16% (0.25-0.18mm), 3% (<0.18mm)) and
the other on fine sand W3 (distribution of grain size classes
7% (0.6-0.2 mm),60% (0.2-0.063 mm), 20% (0.063-0.02
mm), 6% (0.02-0.0063 mm), 3(0.0063-0.0002 mm), 2% (<
0.002mm)). Both obtained in Quartzwerke Frechen, Ger-
many. Relevant parameters in these experiments are the cor-
responding column height, measurement time and the sample
porosity
S
. They are summarized in Table 1.
Table 1 Sample Porosities and Experimental Parameters
Sample Height (cm) Time (min) S
FH31 67 1372 0.36
W3 59 14400 0.36

After having reached complete saturation, the columns
were let to drain by allowing the water to flow out of the
tank. In the special case of FH31, the initial fall of the water
level in the tank was recorded in time (Fig. 2). This is be-
cause the duration of the drainage process in FH31 (about 90
min) is comparable to time the tanks needs to release all wa-
ter (15 min) setting a boundary condition that must be taken
into account for modelling. Afterwards the water is kept at
the same level during the rest of the experimental time. Due
to the fast drainage in FH31, moisture profiles were acquired
before and after the drainage only.

Fig. (2). Pressure head at bottom outside the column.

Another important aspect in the experiment for FH31 is
the drainage dynamics. Since the drainage occurs so fast in
comparison to the time needed to perform a sole moisture
profile, we decided to follow it by maintaining the sensor at
a depth of -22.1 cms during the first 150 min of experimental
time. At this depth, following previous moisture profiles, we
are certain of sensing a considerable drop in moisture. Al-
though this corresponds to read the water content at a sole
point, the data so obtained contains enough information to
characterize the temporal behavior of the drainage com-
pletely. Here the number of NMR acquisitions per moisture
measurements (scans) plays a critical role: It must be high
enough to guarantee a small uncertainty (say 7%) but it
should be made in a time suficiently short to follow reliably
the temporal change in water saturation. In our case, given
the NMR longitudinal relaxation time of water in our sand
(T
1
=800 ms) and a signal-to-noise ratio per echo and per
scan of 0.17 (in FH31), 64 scans with 128 echoes were
enough to achieve the aimed uncertainty (7%) at each point.
In the case of W3 a moisture profile of the water content was
measured each two days.
3. INSTRUMENT AND DATA PROCESSING
Initially perceived as a potential tool for medical research
[14], the NMR slim-line logging tool has been built with a
cylindrical shape. It is an inside-out NMR instrument similar
to some others found in the literature [12]. Fig. (3) shows a
picture of the sensor. Generally speaking, conditions to ob-
serve the nuclear resonance are a constant magnetic field

B0
and a radiofrequency (rf) field

B1 . The latter is generated by

a coil and the former by a magnet body, establishing together
a sensitive volume about 3 mm above the coil (Fig. 4). A
sequence of pulses is applied in the rf field, exciting there-
fore the spins in resonance. Since the inner tube where the
sensor is displaced is only 1 mm thick, the sensitive volume
is 2 mm well inside the soil, avoiding disturbance in the
Low-Field NMR of Water in Model Soils The Open Magnetic Resonance Journal, 2010, Volume 3 65
measurement due to any possible overlapping between the
walls and the sensitive volume. This depth of the sensitive
volume was experimentally checked by measuring the NMR
signal of rubber layers of different thickness. The main body
of the sensor consists of two cylindrical magnets 8 cm long
each (l in the Fig. 4) and 3 cm in diameter (D) and with a
magnetization

M pointed as depicted, separated by a gap g

of 4 mm. Just above the gap lies the rf coil (c). The sensitive
volume is around 30 mm
3
, being its position and thickness
determined by profiles of the magnetic fields

B0 and

B1 ,
the operation frequency (8.8 MHz), the local value of the
magnetic gradient (30 T/m) and the pulse length used
(6 sec) in the pulse sequence [8]. The coil has a dead time
of 20 sec approximately. The NMR spectrometer used was
a Minispec Serie mq from Bruker.
In low field applications, the NMR signal is usually ac-
quired with a Carr-Purcell-Meilboom-Gill (CPMG) [12]
pulse sequence, with pulses separated by a echo time
E
. The
succession of NMR echoes, sequentially acquired, compose
altogether a decaying signal with a effective transverse re-
laxation time (here simply denoted by T
2,eff
). Its decay under
highly inhomogeneous magnetic fields is governed by trans-
lational diffusion of molecules, interactions among nuclear
spins (the proper transversal relaxation) and interactions be-
tween the surface of the solid matrix and the nuclear spins
[13]. In spite of this variety of relaxation mechanisms, aver-
aging over a number of echoes (N
E
) still yields a figure di-
rectly proportional to water content, provided the time span
that corresponds to these echoes (
E
N
E
) is small compared to
T
2,eff
. This can be easily shown by describing the succession
of echoes with these following formulae, being j an index
that labels the echo number (between the 1 and N
E
), n(j) the
noise at the echo j and A
1
the amplitude of the initial echo:

Fig. (4). Main parts of the slim-line NMR logging tool. M labels the
magnetization vector.
S( j) = A1e
( j 1)E
T 2, eff
+ n(J)
If we average over the first N
E
echoes and apply well
known mathematic formulae, we obtain < S >:
< S > =
AO
NE
e
(NE)E
T 2, eff
1
e
E
T 2, eff
1
+ < n > NE
As long as N
E
E
< 0*,*3T
2,eff
, we can approximate the ex-
ponential term to a linear function (e
-x
1 - x) with an uncer-
tainty of 5%. Easy manipulations will lead to the following
equation:
< S >= Ao+ < n >N
E

So it is proven that averaging over the first N
E
echoes has
no further effect on the obtained data than averaging the ran-
dom noise. This way of data processing has the advantage
that for a complete signal of N
E
echoes, we diminish the
noise amplitude to that we had if the experiment were re-
peated NE times. When acquiring noisy signals, this proce-
dure is of not little advantage.
To give an example of this statement with real numbers,
consider the Fig. (5) where data for water in saturated sand is
depicted as it is supplied by the spectrometer. Experimental
parameters in this decay are 256 scans, an echo time of 50 s

Fig. (3). Photo of the slim-line NMR soil sensor.

Fig. (5). NMR signal acquired in saturated sand FH31. T
2,eff
= 28
ms.
and 3000 echoes. With a exponential function fitted to this
whole data, an effective T
2,eff
of 28 ms can be easily ob-
tained. The inset of this figure shows the first 128 echoes,
corresponding to a time of 6.4 ms, a time lower than the
boundary of 30% set by the demonstration given above. So
we can confidently average this first 128 echoes to obtain a
relative measure of the partial saturation in sand. With prior
knowledge of the signal level for completely saturated sand,
the partial saturation in soil can then be calculated by a
linear interpolation. This was the way of processing data for
all the moisture profiles presented here.
4. RESULTS
The evolution of water saturation as a function of depth
is shown in the Figs. (6 and 8) for the sand and the silt col-
umns, respectively. A fact easy to recognize is the big differ-
ence in time evolution: while the FH31 has reached its equi-
librium state in 1.5 hours, W3 does not show any important
difference in saturation during 10 days.

Fig. (6). Moisture profle in a column of model soil FH31.
Height=67 cm.

Four profiles were measured in FH31, one prior to drain-
age (t = 0 min, Fig. 6) and three thereafter (t = 182, 891,
1372 min). Experimental parameters for these measurements
were: Echo time: 50 sec, 256 scans, 3 sec of recycling de-
lay and 128 echoes. The similarity of these three last profiles
reveals that the equilibrium state of the column had been
reached already at the time of the first measurement (182
min). As is well known in soil physics, the profile in this
state corresponds to the retention curve (h) [16]. We used
therefore the profile at t = 891 min for fitting of the retention
curve.
For the dynamical measurement, done by leaving the
sensor measuring at certain depth during drainage as it was
explained before, the results are depicted as open symbols
the Fig. (7). Experimental parameters were the same as for
the moisture profiles with the sole exception of the scans: 64
instead of 256, for reasons already explained. The continu-
ous curve correspond to the numerical solution for perme-
ability at complete saturation K
s
.
The situation with the model soil W3 was completely dif-
ferent. It retained water during 10 days with no measurable
change. Experimental parameters were the same than for
FH31, although the used NE for averaging was 64 instead of
128, as the T
2,eff
for this soil was 20 ms. Given the height of
the column the retention curve of this soil cannot be estab-
lished (Fig. 8). However, a slight drop of 5% from the value
at the bottom can be observed along the profile, correspond-
ing probably to the water loss in the biggest pores.

Fig. (8). Moisture profile in a column of model soil W3. Height=59
cm.

5. MODELING WITH RICHARDS EQUATION FOR
THE FH31 COLUMN
To validate the measurements obtained with this new
method to probe soil moisture, we use the well known Rich-
ards equation (eq. 1) [15, 16]. It establishes the theoretical
framework to model the evolution of moisture profile in the
sand column, where h is the hydraulic head, the partial
saturation, and K
s
the permeability of the soil at complete
saturation. After starting the drainage process the level of the
water mirror that initially was at the top of the column re-
cedes completely to the bottom in about 15 min, setting a
variable pressure head (Fig. 2) at the bottom. This data was

Fig. (7). Moisture drop at depth -22.1 cm during 150 min for FH31.
In color, numerical solution for K
s
=0.62 cm/sec.
Low-Field NMR of Water in Model Soils The Open Magnetic Resonance Journal, 2010, Volume 3 67
taken as lower boundary condition for a numerical solution
of eq. 1.
o
ot
=
o
ox
K()
oh
ox
+1
_
,
1
]
1
(1)
To undertake the simulation of the drainage problem, we
use the Van Genuchten Model [17] for the retention curve
(eq. 3) (where and n are the Van Genuchten parameters)
and the permeability in dependency of the relative saturation
(4), having K
s
the meaning explained in the previous section.
Parameter m equals 1
1
n
and l=0.5 for most soils [15]. With
the relative saturation S
e
defined by eq. 2 in terms of the real
saturation , the porosity s and the residual saturation r; we
obtain a complete set of equations to solve eq. 1. The
HYDRUS code [18] supplies the computational tool to per-
form 1D simulations to model the illustrated drainage proc-
ess.
Several forward simulations were carried out to find the
values of the Van Genuchten parameters and n that would
reproduce the measured data. The best fit was corresponded
to = 0.0268 cm
-1
and n = 9.0. The numerical profile and the
experimental data are depicted in Fig. (9).
Se =

s r
(2)
Se =
1
[1+ (h)
n
]
m
(3)
K(Se) = KsS
e
l
1 1 Se
1
m
_
,
1
]
1
1
2
(4)

Fig. (9). Moisture profile in FH 31 at t=891 min and theoretical
profile using the Van Genuchten model. = 0.0268 /cm
-1
and n =
9.0.

With these gained values for and n, further simulations
were performed with several values of the permeability at
full saturation K
s
, using the temporal change in partial satu-
ration measured at a depth of -22.1 cm for comparison (Fig.
7). Best fit among the numerical and the experimental curve
was found for K
s
= 0.62 cm/min.
CONCLUSION AND OUTLOOK
The instrument has shown its capability of following the
fast drainage process in model soil (FH31). The so obtained
data can be analyzed to extract hydraulical parameters of the
model soil. However, experimental parameter like repetition
time (recycling delay), number of scans and number of ech-
oes must be carefully selected to follow the drainage process
successfully. One interesting remark can be made when ex-
amining Fig. (7): There is some small delay in the moisture
drop that the simulation does not reproduce, perhaps due to
the hysteresis behavior in the retention curve of sand.
What information can be won of this information is still
an open question, however we can see that small features in
the temporal evolution of moisture can be followed with this
instrument. About the Van Genuchten parameters obtained
for FH31, that Kastelanek reports for a sand fraction with a
grain sizes between 150 and 300 m a value of n= 8.35 [19].
The obtained permeability corresponds to 80 % of the value
previously reported [20], measured with Darcy's Law. The
values agree satisfactorily with literature data for a fine sand
of the textural composition like ours, and thus prove the va-
lidity of the NMR sensor.
Work in progress concerns the construction of a sensor
with a larger diameter of 48 mm that allows to measure 6
mm inside the soil at a frequency of 12 MHz. Our short term
goal is to reach a signal to noise ratio per scan and per echo
of 18, similar of the NMR-MOUSE [12] at the same depth.
This would in principle enable us to measure moisture pro-
files in shorter time, to avoid any surface effects due to wall
proximity and to essay external noise suppression tech-
niques, opening the way to a robust instrument which could
be deployed outdoors.
ACKNOWLEDGEMENTS
One of the authors (Sucre) thanks the German Academic
Exchange Service (DAAD) for his Ph. D. grant. Support by
the Deutsche Forschungsgemeinschaft through research pro-
ject TR32 is also grate fully acknowledged.
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ware package for simulating the one-dimensional movement of wa-
ter, heat, and multiple solutes in variably saturated media, version
2.0, U.S. Salinity Laboratory, Agricultural Research Service, Riv-
erside, California USA 2008.
[19] Kastelanek F. Numerical simulation technique for vertical drain-
age. J Hydrol 1971; 14: 213-32.
[20] Pohlmeier A, van Dusschoten D, Weihermueller L, Schnurr U,
Vereecken H. Imaging water fuxes in porous media by magnetic
resonance imaging using D2O as a tracer. Magn Reson Imaging
2008; 27: 285-92.

Received: July 15, 2009 Revised: December 18, 2009 Accepted: December 19, 2009

Sucre et al.; Licensee Bentham Open.



1874-7698/10 2010 Bentham Open
Open Access
MRI in Soils: Determination of Water Content Changes Due to Root
Water Uptake by Means of a Multi-Slice-Multi-Echo Sequence (MSME)
A. Pohlmeier*
,1
, F. Vergeldt
2
, E. Gerkema
2
, H. Van As
2
, D. Van Dusschoten
3
and H. Vereecken
1

1
ICG-4, Research Centre Jlich, Germany
2
Department Biophysics, Wageningen University, The Netherlands
3
ICG-3, Research Centre Jlich, Germany
Abstract: Root water uptake by ricinus communis (castor bean) in fine sand was investigated using MRI with multiecho
sampling. Before starting the experiments the plants germinated and grew for 3 weeks in a cylindrical container with a di-
ameter of 9 cm. Immediately before the MRI experiments started, the containers were water-saturated and sealed, so water
content changes were only caused by root water uptake. In continuation of a preceding work, where we applied SPRITE
we tested a multi-echo multi-slice sequence (MSME). In this approach, the water content was imaged by setting T
E
= 6.76
ms and n
E
= 128 with an isotropic resolution of 3.1mm. We calculated the water content maps by biexponential fitting of
the multi-slice echo train data and normalisation on reference cuvettes filled with glass beads and 1 mM NiCl
2
solution.
The water content determination was validated by comparing to mean gravimetric water content measurements. By co-
registration with the root architecture, visualised by a 3D fast spin echo sequence (RARE), we conclude that the largest
water content changes occurred in the neighbourhood of the roots and in the upper layers of the soil.
Keywords: MRI, root water uptake, root system, relaxometric imaging.
INTRODUCTION
The interplay between root activity and soil functions is
not yet well understood on a local scale, although it belongs
to the most important processes controlling plant growth and
productivity. One reason is the limited observability under
natural conditions, since roots are the hidden half of the
entire plant [1]. Recently, progress has been made by the
development of numerical modelling based on soil physical
principles, where water motion is calculated in a coupled
soil-plant network of water potential differences, hydraulic
conductivities and root system architecture [2-4]. The suc-
cess of such integrated modelling approaches requires ex-
perimental information about the root system architecture as
well as water content distributions under controlled bound-
ary conditions in experimental scenarios. Here, non-invasive
3D tomographic methods are mandatory, since only these
can give an undisturbed look into the soil and the processes
taking place there. Today, three main techniques based on
different physical principles are generally usable, X-ray [5],
neutron [6] and nuclear magnetic resonance tomography
(MRI) [7-10]. The potentials and limits of these methods for
investigating root-soil interactions are a present challenging
topic in soil and plant sciences.
The focus of X-ray CT is on the physical density with
high resolution (some microns), and so this method is highly
sensitive for imaging of the solid phase. On the other hand
the differentiation between water and air turns out to be

*Address correspondence to this author at the Research Centre Jlich, ICG-
4 (agrosphere institute), D-52425 Jlich, Germany; Tel: (49)2461-612795;
Fax: (49)2461-6125; E-mail: a.pohlmeier@fz-juelich.de
tricky. In contrast, MRI is principally capable to image both
root system architecture and water content, since its sensitiv-
ity is based on the presence of water and on the properties of
the medium, in which it is located. Also MRI is capable to
image fluxes or water content changes on different time
scales.
The present work is a continuation of our previous stud-
ies on the use of MRI for the investigation of root water up-
take [10, 11]. There we used a single point imaging tech-
nique, since this is especially convenient for matrices with
short transversal relaxation times as soils at low water con-
tent are [12]. However, single point imaging methods are
time consuming since the resolution scales with the cube of
the repetition time.
In this work we want to elucidate further the potential of
relaxometric imaging for the water content determination
[13]. The major concern for such methods is that with de-
creasing water content the relaxation times decrease strongly
according to the Brownstein-Tarr model [14, 15]. So if one
just uses conventional spin-echo imaging the echo intensity
reflects not only the water content but also the local relaxiv-
ity, and it is not necessarily proportional to the water content.
As model system we have chosen ricinus communis carmen-
cita (castor bean) grown in medium sand. The scenario was
initial saturation and subsequent increasing drought stress
over two weeks by sealing the container and allowing tran-
spiration only via the leaves. Water contents were mapped
by relaxometric imaging using a multi-slice- multi-echo
method (MSME). Additionally, the root system architecture
was imaged by a 3D fast spin echo method (RARE) with
high resolution.
70 The Open Magnetic Resonance Journal, 2010, Volume 3 Pohlmeier et al.
MATERIAL AND METHODS
Plant Setup
10cm high cylindrical Perspex containers with 8 cm in-
ternal diameter were used for all experiments. Each was
equipped with two 0.8 x 15 cm cylindrical marker tubes
filled with glass beads and saturated with 5 mM NiCl
2
solu-
tion as internal water content standard (=0.39) and for
coregistration of the MRI images recorded at different dates.
The containers were filled about 10 cm high with medium
sand (0.2-0.7mm) and a 2cm top layer of peat. Ricinus seeds
have been planted in the peat layer, see Fig. (1). After germi-
nation they grew for three weeks. Then the container was
saturated slowly from bottom and sealed on both sides so
that transpiration proceeded only via the leaves. Three indi-
vidual plants have been grown, of which one is chosen here
for further evaluation of the data.

Fig. (1). Ricinus plant in the container immediately before measur-
ing.

Fig. (2) shows the gravimetrically determined water con-
tent changes with time after saturation and sealing on day-0.
The small vertical arrows indicate the dates of the MRI
experiments.

Fig. (2). Gravimetric total water content in the container after satu-
ration and sealing. The arrows indicate the dates of the MRI ex-
periments.
MRI Hardware & Protocols
All MRI experiments have been performed at the
Wageningen 3T scanner (127.9 MHz, G
max
=0.27 T/m),
operated by a Bruker console. The root architecture was
determined with high spatial resolution using a 3D-RARE
pulse sequence with a turbo factor of 16. T
E
was 6.7 ms for
the first echo and 4.3ms for the subsequent 15 echos, T
R
=
1.5s. The Voxel size was isotropic 0.78mm. Quantitative
water content mapping was performed by a multislice-
multiecho sequence (MSME)[13]. The simplified timing
diagram is displayed in Fig. (3). In contrast to conventional
single-echo pulse sequences it monitors a sequence of many
echoes n
E
for each voxel, addressed by the slice selective
initial 90 pulse, the phase encoding (pe) and the read-out
(ro) gradients. Before further analysis the data, which have
been recorded in the complex space, were phase corrected so
that the information was only in the real part, and the
imaginary part was ignored. This is especially important for
biexponential fitting [16]. The signal intensity for
biexponential decay due to transversal relaxation under the
condition for sufficiently long repetition time is given by
Eq. 1.

Fig. (3). simplified MSME imaging timing diagram. T
E
is echo
time, n
E
the number of echoes, rf, ro, sl, and pe refer to rf-channel,
read-out, phase-encoding, and slice selection gradients, respec-
tively. Further details see text.

s
E E
s
f
E E
f E E
T
T n
S
T
T n
S T n S
, 2
0
, 2
0
exp exp ) (
(1)
S
0
= S
0,f
+ S
0,s
(2)
n
E
and T
E
are the number of echoes and echo time, respec-
tively. S
0,f
, S
0,s
,

T
2,f
, and T
2,s
are the amplitudes and transver-
sal relaxation times of the fast and slow processes, respec-
tively. So, by extrapolation the quantity S
0
= S
0,f
+ S
0,s
is ob-
tained, which is proportional to the spin density and the wa-
ter content in individual voxels. We used the following im-
aging parameters: matrix size 32x32 and 3 mm slice thick-
ness (10x10x9.6cm). Slices were addressed in interleaved
0 2 4 6 8 10 12 14
0.0
0.2
0.4
time (days)
MRI experiments
n
pe

rf
n
E

ro
sl
pe

T
E

MRI in Soils The Open Magnetic Resonance Journal, 2010, Volume 3 71
mode. T
E
= 6.76ms, n
E
= 128, T
R
= 30s. The water content
maps are obtained by calibration of the 3D amplitude maps
on S
0
of the voxels of the calibration tubes. All data evalua-
tion steps were performed by the IDL programming system.
Fig. (4) shows some exemplary horizontal and vertical
slices through the ricinus container. Plotted are the intensity
of the first echo and relaxation curves of selected voxels in-
dicated by the yellow arrows. Although the resolution is lim-
ited to 3 mm, we observe structural details in the container.
In Fig. (4a) the yellow arrow points to the calibration tube.
In Figs. (4b) to (4e) roots are clearly resolved, as well as the
peat layer in the upper part of the container, which is much
more heterogeneous than the sand packing below. Also the
signal intensity is much higher. In Fig. (4d) a root is indi-
cated and in Fig. (4e) the arrow points to the onset of the
shoot inside the peat layer. In the lower parts of the subfig-
ures relaxation curves in selected voxels are shown for a) in
the marker, b) and c) in bulk soil on day-1 and day-6 after
saturation, d) is from a root voxel and e) from a shoot voxel.
One should take into account that the voxel size is greater
than the dimension of the roots, so that the intensity is com-
posed of both signals from root and the immediately sur-
rounding soil. This partial volume effect is responsible for
the lower signal intensity in the root-voxel compared to the
shoot voxel, since the shoot is much thicker than the roots.
In most voxels bimodal transverse relaxation was ob-
served, so Eq. 1 was fitted to all voxels. From that we ob-
tained amplitude, T
2,f
and T
2,s
maps. This is exemplarily
shown for 30 vertical slices in Fig. (5) for the plant on day-1.
The amplitudes are quite uniform over the sand packing,
with exception of the slices 2 and 3 from bottom. Clearly
distinguishable is the peat layer in the upper 2 cm. It is char-
acterized by high amplitudes reflecting high water contents
and by short fast transverse relaxation times T
2,f
, which are in
the range between 3 and 8 ms, whereas the slow relaxation
time T
2,s
is about 80ms and comparable to the range of the
sand packing. The second feature is the inner and outer
marker tubes in images 16 to 18 and 26 to 30, respectively,
characterized by moderate amplitudes and long relaxation
times (T
2,f
20ms, T
2,f
200ms). Also voxels including roots

Fig. (4). Exemplary MSME-MRI images of selected slices of the ricinus container. a) axial slice near bottom at day-1, b) and c) axial slices
near top on day-1 and day-6, resp. d) and e) vertical slices showing roots and shoots. The arrows indicate individual voxels for which the T
2

decay curves are shown in the lower parts of the subfigures.
a) b) c)

d) e)
are visible in the amplitude maps of the central vertical
slices, whereas they are not clearly identifiable in the relaxa-
tion time maps. However, it should be noted that in the areas
with highest root densities, e.g. slices 17 and 18, both fast
and slow relaxation times are faster (T
2,f
8ms, T
2,f
50ms),
than in the outer regions of the container (T
2,f
14ms, T
2,f

80ms). The nature of the fast and slow relaxation time is not
yet clear. Assuming that the Brownstein-Tarr model is valid,
transverse relaxation is accelerated in small pores due to
frequent wall contacts of diffusing water molecules in the
neighbourhood of paramagnetic centres. Second, transverse
relaxation is accelerated by diffusion in internal gradients
[14]. This might explain the frequently observed bimodal
relaxation in natural porous media, but this requires more
detailed investigations, which exceed the frame of this work.
The central aim of this work is the determination of water
content changes due to root system activity. As the next step
we have calculated the water content (x,y,z) by calibration
of the total amplitude maps on the marker tubes with =
0.39. This evaluation procedure is validated by comparing
the average gravimetric water contents of the plant container
with the MRI determined water content.
Fig. (6) shows that MRI-water content profiles, which
have been obtained by integration over horizontal agree well
with the average gravimetric water contents. This proves the
validity of the entire measurement and evaluation procedure.
Examples of the water content map for slice 19 on day-1 and
day-6 are shown in Fig. (7). One can clearly recognize the
roots and the peat layer on the top. The soil appears quite
homogeneous with some enrichment of water near the bot-
tom. On day-6 the whole system is much drier, the average
water content in the sand is about = 0.05. The top peat
layer is also much drier, so that the onset of the shoot be-
comes visible. A striking point is that the voxels containing
roots also appear much drier (blue colour vs. red colour on
day-1), although one might expect that the roots do not loose
water in significant amounts unless the plant is wilted. So
they should appear white just like the shoot. The reason for
this is that the voxel size is with 3 mm significantly larger
than the root diameter, so the lower intensity is due to a par-
tial volume effect; one observes average water content com-
posed of root and surrounding soil.

Fig. (6). Vertical water content profiles of the ricinus-root soil sys-
tem, calculated by integration over horizontal slices from the water
content maps.

For a comparison of the root system with the water con-
tent maps we have also recorded 3D images of only the roots
by MRI with a 3D RARE sequence. This is presented in Fig.
(8), where voxels with intensities above a threshold of 25%
of the maximum are enclosed by an isosurface. The peat
layer is cut off.

Fig. (5). Total amplitude S
0
, T
2,f
and T
2,s
maps obtained for the ricinus container on day-1 by fitting of Eq. 1 to the relaxation curve in all
voxels. Shown are 30 from 32 vertical slices, FOV is 10x10cm, matrix size 32x32. The slices are enumerated 1 to 30 from top-left to bottom-
right.
S
0f+s
0 400
800au
T
2,f
0 25 50 ms
T
2,s
0 100 200 300 ms
0
20
40
60
80
100
0.0 0.1 0.2 0.3 0.4 0.5 0.6
mean water content
h
e
i
g
h
t

f
r
o
m

b
o
t
t
o
m

/

m
m
day 6 day3 day1
MRI in Soils The Open Magnetic Resonance Journal, 2010, Volume 3 73

Fig. (7). Selected water content maps of the ricinus-root soil sys-
tem. Shown is a central vertical slice on day1 and day6 after satura-
tion and sealing.

Fig. (8). 3D MRI-RARE image of the ricinus root system on day-1.
The scales are voxels, voxel size is 0.078mm, matrix size
128x128x128, FOV 10x10x9.6cm.

Beneath the roots spots are also visible, which do not be-
long to roots but are sites with locally high water content.
For an easier comparison to the water content maps Fig. (9)
presents an excerpt from the 3D image in Fig. (8), which
corresponds to the vertical slice shown in Fig. (7).
The maximum width of the main roots is 3 voxels, i.e.
2.3mm which is much smaller than the voxel size for the
water content maps. Going back to Fig. (6) one can see that
for example the central root appears about 3 voxels wide, i.e.
about 9mm. Since this is much larger than the root thickness
itself this strongly indicates wetter zones around the roots.
Such behaviour has also been observed by others [17, 18].
The final step of the evaluation is the calculation of water
content difference maps in order to see where the root sys-
tem has taken up water preferentially. In Fig. (10) the root
system architecture is overlaid with four axial slices of the
water content differences between day-1 and day-6. The wa-
ter content change was greatest in the upper central part of
the soil core, whereas near bottom if remained wetter. Also
in the neighbourhood of the roots greater changes of the wa-
ter content is observable. This indicates that the hydraulic
conductivity is decreased so far that the water uptake by the
roots is not yet compensated instantaneously by flux from
more distant regions.

Fig. (10). ricinus-root soil system. Difference of water content
between day-6 and day-1, overlayed by the root system, obtained
from the RARE MRI measurement. (The long cylinders in the left
half are the marker tubes).

CONCLUSIONS
MRI was used for both imaging of the root system archi-
tecture and the water contents in a three week old ricinus
Fig. (9). Central vertical slice of the MRI-RARE image of the
ricinus root system.
: 0 0.5
day-1 day-6

8 cm

grown in natural sand. For the determination of the water
content it is advantageous to use a relaxometric multi-echo
method since this yields relaxation time maps as well as am-
plitude maps, from which water contents can be obtained by
calibration on internal reference samples. The results of these
investigations agree with those we have obtained in a previ-
ous work by the application of a single point imaging
method (SPRITE) [10]. But in contrast to that MSME is
faster, since the total measurement time single point imaging
methods scales with the cube of the resolution, echo methods
only with the square. The second advantage of MSME is the
determination of T
2
maps which reflect local soil properties,
whereas SPI determines T
2
*
, which is mainly controlled by
magnetic field inhomogeneities. On the other hand multi-
echo imaging requires T
2
relaxation times greater than some
milliseconds, since the minimum T
E
is about 1.5 ms. Since in
many natural soils T
2
can be faster, multi-echo imaging
would show no signal, so that single point imaging must be
applied.
By the analysis of the water content maps we found that
in the neighbourhood of the roots zones with increased water
content exist also when the total water content decreased to
< 0.05. Anyway, the water content change in some these
areas was greater than in the bulk soil, which can be ex-
plained by initially increased water content. The next step of
the analysis will be soil physical modelling of root water
uptake processes, which requires a continuous representation
of the root system (skeletisation). For this purpose the qual-
ity of the root system imaging should be improved in terms
of a better discrimination between soil and roots and the
closing of apparent gaps along the roots strands. This could
by performed by either other imaging methods or image
processing techniques.
ACKNOWLEDGEMENTS
The authors are grateful to EU for funding the NMR ex-
periments at the Wageningen NMR Centre (WNMRC07-
001), to the DFG (SFB TR-32, PO-746-2/1) for financial
support, and Beate Uhlig, ICG-3, Research Centre Jlich for
growing the plants.
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Received: July 15, 2009 Revised: November 17, 2009 Accepted: November 20, 2009

Pohlmeier et al.; Licensee Bentham Open.



1874-7698/10 2010 Bentham Open
Open Access
Effect of RF Field Inhomogeneity and Sample Restriction on Spectral
Resolution of CP/MAS-
13
C NMR Spectra of Natural Organic Matter
Anne E. Berns*
,1
and Pellegrino Conte
2

1
Institute of Chemistry and Dynamics of the Geosphere, ICG-4: Agrosphere, Forschungszentrum Jlich GmbH, 52425
Jlich, Germany
2
Dipartimento di Ingegneria e Tecnologie Agro-Forestali (DITAF), Universit degli Studi di Palermo, v.le delle Scienze
13, edificio 4, 90128 Palermo, Italy
Abstract: It is well known that the induced B
1
magnetic field in an NMR coil is inhomogeneously distributed. However,
this issue has so far received little attention in the field of environmental NMR. As this research field often aims at quanti-
tative results as well as relaxation phenomena, the repercussions of such inhomogeneity on peak integrals and relaxation
times need to be taken into account.
The objective of the present study was to test standard recording conditions on different sample positions in an NMR coil
in order to determine the effect of the RF field inhomogeneity on the spectrum of a molecularly complex humic material
and on some standard molecules of known structure and conformation. To this end, we measured the peak integral and
signal half-height width of constant sample amounts at different heights in the rotor. In addition, the effect of sample posi-
tion in the rotor on T
1
H and T
2
C relaxation times was determined.
We showed that the response profiles of different chemical groups are not necessarily comparable to each other and that
spectra of natural organic matter can change when confined to different regions of the coil. Furthermore, the relaxation
measurements revealed that T
1
H and T
2
C relaxation times are position-dependent. Finally, the application of sample res-
triction to the homogeneous region appeared very promising for enhancing the resolution of spectra of complex mixtures.
Keywords: CP/MAS
13
C-NMR, RF field inhomogeneity, physical sample restriction, natural organic matter, signal response
profile.
INTRODUCTION
Cross-polarization magic-angle spinning (CP/MAS)
13
C-
NMR spectroscopy has become a popular technique for
studying natural organic matter (NOM) [1, 2] both in bulk
soils and sediments [3] or when NOM is isolated from
environmental matrices [4, 5]. However, this technique
involves a number of pitfalls.
It is a well-known fact that the magnetic field induced by
a radio frequency (RF) in a finite coil rapidly drops at both
ends of the coil [6], resulting in an inhomogeneous distribu-
tion of the B
1
field in NMR spectroscopy [7]. This inho-
mogeneity, inherent in all commercial standard solenoid
coils, hampers, for example, quantitation of Bloch decay and
CP experiments using spin counting [8, 9], where a carefully
weighed quantity of an intensity standard [10] is added to a
sample and peak integrals are compared. Restricting the
sample to the homogeneous region of the solenoid coil is the
easiest and most practicable way to avoid an inhomogeneous
B
1
field. However, the usable volume might be small and
samples containing nuclei with low NMR sensitivity, be-
cause of either low magnetogyric ratio values or low natural
abundance, may quickly reach the limits of feasibility.

*Address correspondence to this author at the Institute of Chemistry and
Dynamics of the Geosphere, ICG-4: Agrosphere, Forschungszentrum Jlich
GmbH, 52425 Jlich, Germany; Tel: +49 2461 615656; Fax: +49 2461
612518; E-mail: a.berns@fz-juelich.de
Several optimized winding geometries such as coils with a
variable pitch [7, 11, 12] or a variable wire width [13] have
been proposed in order to enhance the homogeneous volume
of solenoid coils. Furthermore, a number of selective pulse
sequences and gradient techniques for spatial localization
and sample restriction have been developed [14-20].
In addition to the inherent B
1
inhomogeneity in solenoid
coils, RF profiles of different frequencies, e.g.
1
H and
13
C,
are not necessarily symmetric about the coil centre as re-
ported by Paulson et al. [21]. This phenomenon of wave-
length effect, which results in a misalignment of the physical
centre of the RF field, was first reported by Stuhlman &
Githens in 1932 [22] and becomes experimentally important
once the coil wire length reaches about 10% or more of the
free space wavelength of the applied RF field [21]. This is
especially of concern for the
1
H field as this critical coil
length can be easily reached even at lower frequencies. A
slight field centre misalignment of the
1
H field is usually
counteracted by using a variable amplitude or ramped CP
transfer [23], which sweeps through a range of (usually)
1
H
RF field amplitudes. For severe misalignments of two or
three RF fields in a double or triple resonance probe, Paulson
et al. [21] developed a balanced series resonant circuit,
which splits the capacitance across the inductor instead of
using a resonant circuit with only one capacitance. Unfortu-
nately, this hardware change is not practicable in every rou-
tine NMR lab.
76 The Open Magnetic Resonance Journal, 2010, Volume 3 Berns and Conte
Magic-angle spinning in combination with cross-
polarization adds to the problem of field inhomogeneity as
dipolar interactions become time-dependent. In a non-
spinning sample, the most efficient magnetization transfer is
reached when the B
1
field mismatch parameter =
1I

1S

between two spin systems I and S is close to zero. In a sam-
ple which is spun at a rate
r
the most efficient transfer oc-
curs when = n
r
(sideband matching condition) [21,
24]. At high spin rates, when the mismatch parameter be-
comes a considerable fraction of the RF field nutation rate,
i

B
1
, the necessity of a homogeneous RF field becomes very
important. This requirement is even more stringent at an op-
erating frequency of 800 MHz or more when MAS rates
reach or exceed dipolar couplings of
1
H-
13
C or
1
H-
15
N [21].
Spectral resolution depends upon the length (N) of the
transform interval: N = F/f, where f is the smallest fre-
quency difference which can be distinguished at a given
sampling frequency F. The frequency difference f depends
on the signal width, which, in turn, is influenced by the re-
laxation time of the sampled signal [25, 26]. Relaxation
times are affected by the external magnetic field and the ap-
plied RF field sent to the sample through the transmit-
ter/receiver coil. As already described above the RF field
homogeneity throughout this coil is a major factor affecting
the NMR sensitivity and the resolution of the measurements.
The objective of the present study was to check standard
recording conditions on different sample positions in an
NMR coil in order to determine the effect of the RF field
inhomogeneity on the spectrum of a molecularly complex
humic material and on compounds of known structure and
conformation. To this end, we performed CP/MAS-NMR
experiments where constant sample amounts were moved
through different heights in the rotor. At each height the
peak integral and signal half-height width were measured
and used as indicators of sensitivity and spectral resolution.
In addition, the effect of sample position on T
1
H and T
2
C
relaxation times was determined.
Standard Materials
Sodium dodecylsulfate (SDS) was purchased from
Sigma-Aldrich
(Steinheim, Germany) in purum quality and

glycine was purchased from Merck (Darmstadt, Germany) in
pro analysi quality. The standard materials were used with-
out further purification. Pure quartz was purchased from
Merck (Darmstadt, Germany) in pro analysi quality and
ground in a planetary mill (PM 400, Retsch, Haan, Germany)
to fine powder.
Humic Acid
A humic acid from the A horizon of an andosol from the
caldera of Vico (near Rome, Italy) was extracted, purified
and characterized as reported in Cozzolino et al. [27]. The
ash content was below 3 % and the Fe/C ratio 1, hence the
CPMAS
13
C-NMR spectra were not affected by paramag-
netic impurities [28].
The HA was analysed to determine its elemental compo-
sition (C, H, N) in a Fisons Interscience EA1108 elemental
analyser. The elemental composition corrected for the ash
content (< 3 %) was as follows: C 56.8 %, H 4.5 %, N 5.2 %.
The humic acid was also analysed by atomic-absorption
spectrometry (AAS) in order to verify the presence of poten-
tially paramagnetic species (Fe, Mn, Cu) in the ash content.
The AAS analyses were performed on a PerkinElmer Ana-
lyst 700 with an instrumental sensitivity of 0.1 mg kg
-1
. Ali-
quots of the humic acid (50 mg) were boiled in a ni-
tric/perchloric acid solution until complete mineralization of
the organic matter and dissolution of the inorganic ashes was
obtained. The solutions were transferred to 20-mL volumet-
ric flasks and analysed by AAS. Negligible traces of
paramagnetic metals were found.
General Solid-State NMR Conditions
A 7.05 T Varian UNITY INOVA (Varian Inc., Palo
Alto, CA, USA), equipped with an Apex HX wide-bore
probe operating at a
13
C frequency of 75.4 MHz, was used to
acquire the
13
C-NMR spectra. The samples were packed in 6
mm zirconium rotors with Teflon
bottom and top spacers

and Vespel
drive tips. The temperature was kept constant at

25.0 0.1 C. Magic-angle spinning was carried out at 7500
1 Hz. Repetititon times were 3s for glycine and 35 s for
SDS. The spectra of glycine and SDS were recorded with a
1
H RF field strength of 65.3 kHz with a ramp of 7 kHz.
CP/MAS spectra of Vico HA were acquired with a
1
H RF
field strength of 40.4 kHZ and a ramp of 15.3 kHz to account
for inhomogeneities of the Hartmann-Hahn condition [2, 21,
23, 24]. In both cases the matching
13
C and
1
H RF fields
were determined in separate (non-ramped) experiments by
fixing the
13
C RF field and arraying the (non-ramped)
1
H RF
field. Decoupling was done using a TPPM sequence with a
1
H field strength of 54 kHz, a phase of 8 and a pulse length
of 9.3 s.
VNMRJ software (Version 1.1 RevisionD, Varian Inc.,
Palo Alto, CA, USA) was used to acquire all the free
induction decays (FID). Spectra elaboration was performed

Fig. (1). Pencil
rotor and dimensions of manufacturer-determined

sample volume.
Effect of RF Field Inhomogeneity The Open Magnetic Resonance Journal, 2010, Volume 3 77
by Mestre-C software (Version 4.9.9.9, Mestrelab Research,
Santiago de Compostela, Spain). All the FIDs were
transformed by applying first a 4 k zero filling and then an
exponential filter function with a line broadening (LB) of 20
Hz. Fully automatic baseline correction using a Bernstein
algorithm was applied for baseline corrections [29].
Sample Positioning in the Rotor
A constant sample amount was incrementally moved in a
volume with 2 mm fill height through different heights in the
rotor to record the spectra of the two standard substances at
different positions in the rotor (Fig. 1). At every height the
peak integral and the half-height width of the signals were
determined as indicators of sensitivity and spectral resolu-
tion. Furthermore, relaxation times of selected volumes were
determined. The amount of sample needed to fill a volume of
2 mm fill height was 32 1 mg for glycine (= 0.43 mmol)
and 26 1 mg for SDS (= 0.10 mmol). In a first set of ex-
periments with SDS and glycine, the bottom and top parts
were filled with fine quartz powder with a constant density
of 26 1 mg/mm. This was done to ensure a constant total
weight of the rotor (3.6 0.01 g) and avoid possible height
differences due to the lifting of the rotor during spinning.
The replacement of a defective preamplifier, although
quickly discovered, during the first set of experiments,
caused slightly different recording conditions for SDS and
glycine so that we decided to verify the reproducibility of
these experiments. The repetition of the experiments with
SDS and glycine was done with purpose-built boron nitride
inlets to facilitate the positioning of the sample and ensure an
even better weight constancy of the rotor. The results re-
ported are from this second set of experiments, which con-
firmed the first set. The correct position of the sample in the
rotor was determined with the help of a light table and a
template. The total possible sample volume was given by the
Teflon
top and bottom spacers provided by the manufac-

turer matching the coil geometry. The bottom limit of this
volume was defined as position 0 mm and the top as 14 mm
(Fig. 1). The position of a sample is indicated through the
position of the centre of the sample in question. For example,
a sample filling a volume from 2 to 4 mm height is indicated
by the position at 3 mm.
After determination of the homogeneous region, experi-
ments were conducted with centre, bottom & top and fully
filled rotors. In this case centre filled refers to samples
where ground quartz powder (or BN inlets) was filled from 0
to 5 mm and from 11 to 14 mm and the sample occupied the
volume from a height of 5 to 11 mm. The sample position
was checked with the help of a light table and a template. In
bottom & top filled rotors, the sample occupied the lower
and top part of the rotor, while the centre was filled with
ground quartz powder (or BN inlet).
CP/MAS and Relaxation Experiments
T
1
H relaxation was determined with the sequence
displayed in Fig. (2A) with an array of delay times (d3) from
0 to 2 s for glycine and an array of 0 to 11 s for SDS. A spin
echo sequence with an array of tau from 0 to 5333 s was
performed to determine the T
2
C relaxation time constant
(Fig. 2B). The fitting procedures for the relaxation curves
were done with OriginPro 7.5 SR6 (Version 7.5885,
OriginLab Corporation, Northampton, MA, USA). The
errors reported are obtained from the fitting procedure.
The CP/MAS
13
C-spectra were evaluated for their peak
integrals and widths at half-height. The determined integrals
and widths at half-height were normalized to position 8.
In the 7.05 T instrument used for the present study, the
1
H radio frequency of 300 MHz corresponds roughly to a
free space wavelength of 1 m. Hence, the resulting critical
coil wire length, where wavelength effects are to be expected
[21, 22], is 10 cm. The Apex Pencil
probe utilized for the

present study is equipped with a 5-turn coil with an inner
diameter of 6 mm and a wire thickness of 1 mm. Thus, the
coil wire length is approximately 11 cm, excluding the lead
lengths. The RF of 75 MHz for the
13
C nucleus corresponds

Fig. (2). T
1
H pulse sequence (A) and T
2
C echo pulse sequence (B).
to approximately 4 m, i.e. a critical coil wire length of 40
cm. Hence, for the
1
H RF field a slight centre misalignment
can be expected as the coil wire length amounts to a mini-
mum of 11 % of the applied wavelength. A rapid measure-
ment of
1
H and
13
C-integrals on a 2-mm-thick adamantane
sample confirmed that the centres of both RF profiles were
not perfectly aligned (data not shown). Therefore, a ramped
1
H field to counterbalance this misalignment must be ap-
plied.
To avoid RF field nutation rates, which match rates
equivalent to the MAS rate,
r
, or twice as great, 2
r
, (usu-
ally conditions which destroy spin-locked magnetization
during cross-polarization through recoupling of chemical
shift anisotropy), an RF nutation rate exceeding 5
r
is rec-
ommended [21, 24]. A spin rate of 7.5 kHz and
1
H RF fields
of 40.4 kHz and 65.3 kHz, as reported in Materials and
Methods, were chosen for the present study. These condi-
tions ensured that the RF field strengths exceeded the rec-
ommended minimum RF field strength of 5
r
(i.e. 37.5 kHz)
so that no negative effects on the spin-locked magnetization
were to be feared.
It should be noted that pulse widths and matching condi-
tions were set with completely filled rotors and, as a conse-
quence, sensitivity profiles exhibit less dependence than they
would if the parameters were optimized on a centre-filled
rotor [8].
The CP/MAS spectra of glycine and sodium dodecyl sul-
fate recorded at different positions within the manufacturer-
defined volume are displayed in Fig. (3). The detection-
sensitivity profiles clearly show that the signal integrals are
strongly dependent on the position of the sample in the rotor.
Only in a small region in the centre of the rotor is the signal
response reasonably consistent.
Figs. (4A) and (4B) show the normalized signal integrals
of these CP/MAS spectra. The signal integrals are normal-
ized to position 8, the centre of the determined homogeneous
region. The sensitivity profile of the glycine methylene
group displays a smooth curve, while the curve of the COOH
group has two outliers in positions 8 and 9. In the profile of
SDS, however, only the integrals of signal C2-10 are consis-
tent. Considering the molar amounts present in the rotor, it
becomes clear that signal C2-10 corresponds to 0.90 mmol

Fig. (3). Response profiles of glycine (A) and SDS (B).
of C, the two signals of glycine to 0.43 mmol C each and the
remaining signals of SDS correspond to only 0.10 mmol C
each. These reduced signals result in a larger error in the
evaluation of the spectrum. Nonetheless, the general shape of
the profile (as well as the profile of the first experiment, not
shown) follows that of glycine with severely reduced signal
integrals in the outer regions; the lower positions being
worse than the upper regions.
As already stated by Campbell et al. [8], sensitivity pro-
files in cross-polarization experiments (as used in the present
study) should be expected to display a much less pronounced
drop at the end of the coil. In fact, the inhomogeneous ampli-
tudes of the
1
H and
13
C RF fields are counterbalanced
through the Hartmann-Hahn signal enhancement as long as
the ratio of the
1
H/
13
C RF field levels is maintained (which is
given by the use of a ramped
1
H RF field). Campbell et al.
[8] mention two factors which need to be considered in such
position-dependent sensitivity profiles in Bloch decay and
cross-polarization experiments. The first factor is the varia-
tion of the RF field magnitude, which results in different
pulse flip angles for different regions in the coil. The second
factor is the variation of the curvature of the B
1
field, which
produces magnetization elements that are directed out of the
detection plane. Due to the symmetry of the coil, these non-
planar magnetization elements exist in opposing pairs which
cancel each other out. Hence, not all of the generated mag-

Fig. (4). Sensitivity (A and B) and resolution profiles (C to F) of the Apex Pencil
probe for
13
C-CPMAS spectra of glycine (A, C and E)
and SDS (B, D and F) (all normalized to position 8 or the central region, respectively).
netization is observed. According to Campbell et al. [8],
these directional inhomogeneities are the main reason for the
spatial dependence of the sensitivity profiles in cross-
polarization experiments (as the inhomogeneous amplitudes
of the
1
H and
13
C RF fields should be counterbalanced
through the Hartmann-Hahn signal enhancement).
In the case of the present study where the samples were
high-rate-spun (Campbell at al. [8] studied non-spinning
samples), a further factor needs to be considered. As the RF
field amplitudes drop at the end of the coil, they may reach a
value where the condition RF field strength > 5
r
is no
longer met, and they may in contrast approach a condition
where the RF field nutation rates
i
B
1
match the MAS rate
r
or 2
r
, thereby destroying the spin-locked magnetiza-
tion during cross-polarization. Unfortunately, no equipment
was at hand for a direct mapping of the RF field strengths.
Furthermore, an exhaustive discussion of the effect of MAS
on CP is already available in several high-quality papers [21,
24] and would go beyond the scope of the present paper.
For quantitative evaluations it is important to know
whether the reduction in signal integral is distributed uni-
formly all along the NMR signals of each chemical group. In
the case of glycine at the lowest positions of the manufac-
turer-defined region, the response decrease of the methylene
group is stronger than that of the carboxyl group. The signal
response profiles of the SDS signals are harder to interpret as
the signal response is very scattered (even in the homogene-
ous region determined), but in the lower region it can be seen
that the signal of the methylene groups C2-10 tends to be
disproportionately reduced as can be seen in the profile of
glycine.
The line widths of the signals are also position-dependent
as displayed in Figs. (4C) and (4D). The normalized display
(to position 8 as in the signal response profiles) shows that
the widths at half-height of the different groups broaden to a
different extent outside the central homogeneous region. The
line width of the glycine methylene group, already broader
by nature through its faster relaxation, shows a stronger in-
crease (factors 2.6 at positions 3 and 12) in its line width
than the carboxyl group (factors 1.6 and 1.4 at positions 3
and 12). Also in the case of SDS, the methylene signals (C2-
10 and C11) are the ones with a larger enhancement of the
width at half-height outside the central region, while the line
width of the methyl group (C12) is hardly perturbed at all.
The width at half-height of the methylene signal closest to
the sulfate group is already quite broad in the centre regions,
and in most of the outer regions the signal width cannot be
measured. The normalized widths at half-height determined
on fully, centre and bottom & top filled rotors are shown in
Figs. (4E and 4F). The signal widths in the bottom & top
filled rotors were elevated similar to the widths in the incre-
mented experiments, whereas the methylene signal of gly-
cine was too broad to be reasonably measured. The widths in
the fully filled rotors were in between the widths of the cen-
tre and bottom & top filled rotors as expected.
The line width is determined by the overall decay rate of
the transverse magnetization T
2
*. The latter, in turn, is af-
fected by both homogeneous (the transverse relaxation rate
T
2
) and inhomogeneous contributions (called inhomogene-
ous broadening and often denoted T
2
). The transverse re-

laxation rate T
2
is a fundamental property and is influenced
by the type of molecule, its physical environment and its
motion in this environment. Inhomogeneous broadening re-
sults from non-uniform magnetic fields across the sample. A
poorly shimmed probe head, for example, causes spins to
experience different Larmor frequencies in different parts of
the sample due to an inhomogeneous B
0
field. The homoge-
neous contribution can be distinguished from the inhomoge-
neous part through a simple echo sequence. As the latter is
due to spins precessing at different frequencies throughout
the sample over time they get out of step and eventually can-
cel each other out, resulting in a decay of the overall mag-
netization. This simple spin dephasing can be reversed by
applying a 180 pulse after an evolution time and recording
the signal after another period, at which point the phases
will have realigned. The homogeneous part cannot be re-
versed as it is a true relaxation process, i.e. an approach to
equilibrium [25, 26]. Measurement of the T
2
C relaxation
times are shown in Figs. (5A and 5B). It can be seen that
especially the longer relaxation times of the COOH group of
glycine and the methyl group of SDS are strongly influenced
by the position of the sample. The application of an echo
sequence where decoupling started only with the recording
of the signal revealed that dephasing during the echo periods
is sensitive to decoupling. Without decoupling being turned
on during dephasing the determined relaxation times showed
no position dependence and were extremely short by reason
of magnetization loss due to coupling (data not shown). It is
hence likely that the higher T
2
C relaxation times determined
in the centre of the rotor arise from more efficient decou-
pling of the sample, due to the increased homogeneity of the
RF pulses in this region. Calculation of the overall decay rate
of the transverse magnetization T
2
* from the determined
widths at half-height revealed a large discrepancy between
the overall decay rate and the T
2
C relaxation, e.g. a T
2
C of
25 ms and a T
2
* of 6.4 ms for the COOH group of glycine.
Hence most of the overall decay rate of the transverse mag-
netization is due to inhomogeneous contributions. As the
transverse relaxation takes place after the application of the
B
1
field the inhomogeneities influencing this factor must
come from the B
0
field. Inhomogeneities in the static mag-
netic field are counterbalanced as effectively as possible by
the shimming procedure. However, the simple presence of
the coil in the magnetic field and the pulsed application of
the B
1
field lead to disturbances in the B
0
field which are
hard to counterbalance completely. Furthermore, the shim-
ming is optimized to the line shape of adamantane, i.e. while
the B
1
field is active. The disturbances which the coil pro-
duces in the B
0
field are strongest at the edges of the coil and
therefore the line widths increase in these regions. In the
main, it must be noted that the determination of T
2
C times in
the fully filled rotor obviously underestimates the relaxation
times.
Measurement of the longitudinal relaxation T
1
H shows a
longer T
1
H in the centre position than in the outer regions
(Figs. 5C and 5D). This is due to the fact that as the RF field
is not homogeneous, the 90 pulse is not perfect throughout
the sample. Hence the outer regions of the sample experience
a pulse which differs from 90 resulting in a lower magneti-
zation from which the system needs to return to equilibrium.
The centre position is closest to the true 90 pulse and hence
has the longest relaxation time. It can be seen that measure-
ments on a fully filled rotor also lead to an underestimation
of the T
1
H relaxation times.
In Fig. (6) the influence of physical sample restriction on
the resolution of a spectrum of a humic acid is shown. All
three spectra were recorded as having a similar signal-to-
noise ratio and were graphically normalized to the carboxyl
signal at 172 ppm. It can be seen that the relative integrals of
some signals in the HA spectrum change, which leads to
differences in the peak integrals ranging from 1 to 3 %. The
main regions influenced are the aliphatic and O/N-alkyl re-
gions (0-45 and 45-90 ppm). The signal in the aliphatic re-
gion of the bottom & top filled rotor (blue spectrum) is
shorter and broader than the signal resulting from the central
region (red spectrum) and that in the spectrum of the fully
filled rotor (black spectrum). Most interesting is the fact that
in the O/N-alkyl region the resolution of the signals is en-
hanced. Between 45 and 65 ppm the blue spectrum of the
bottom & top filled rotor shows only one broad signal,
whereas in the red spectrum of the centre filled rotor three
peaks can be distinguished. The black spectrum of the fully
filled rotor merges both features and is less resolved than the
red spectrum, but better resolved than the blue spectrum. The
more strongly enhanced resolution of the O/N-alkyl region
can also be seen in the spectra of glycine (Fig. 4C), where
the signal half-height width of the methylene group is re-
duced by a factor of 2.6 when moving from the outer to the
central region. As already stated for the T
2
C measurements,
the increase in resolution probably arises from a more effi-
cient decoupling in the centre of the rotor.
Although the physical reduction of the sample to the cen-
tral region requires longer measurement times to reach a
good signal-to-noise ratio, the resolution enhancement
achieved can be of importance when the interactions of hu-
mic acids with labelled chemicals [30] or changes in a spe-
cific region are monitored [31].
CONCLUSIONS
Although RF field inhomogeneity is a subject well studi-
ed by NMR scientists in chemical and physical research
groups, the issue has so far received little attention in the
field of environmental NMR. As this research field often
also aims at quantitative results as well as relaxation pheno-
mena, the repercussions of the position dependence of the
peak integral and of relaxation times on such NMR measu-
rements need to be taken into account.
The present study shows that the response profiles are not
necessarily similar for different chemical groups and hence
spectra of complex organic matter can change when confined
to different regions of the coil. It must be pointed out that the
work presented in this paper was done on a 6 mm probe he-
ad. The described effects might be smaller in smaller rotors
or in coils with different geometry. We therefore recommend
that the signal response profile in the coil should be checked,
for example by the proposed procedure, and the sample
should be restricted to the determined homogeneous region

Fig. (5). T
2
C (A and B) and T
1
H (C and D) relaxation rates of glycine (A and C) and SDS (B and D).
when quantitation is required as also Bloch decay experi-
ments are sensitive to coil sample position. The same applies
to relaxation measurements as relaxation times are also posi-
tion-dependent. Furthermore, the application of sample res-
triction to the homogeneous region appears very promising
for enhancing the resolution of spectra of complex mixtures.
ACKNOWLEDGEMENTS
The authors thank Forschungszentrum Jlich GmbH
(Germany) for financing PC as a visiting scientist at the
NMR centre of the Institute of Chemistry and Dynamics of
the Geosphere, Institute 4: Agrosphere. Many thanks are also
due to Dr. Andreas Pohlmeier for helpful discussions on
relaxation phenomena. The Language Services of
Forschungszentrum Jlich are also gratefully acknowledged
for revising the English of the manuscript.
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Berns and Conte; Licensee Bentham Open.


1874-7698/10 2010 Bentham Open
Open Access
Interaction of a Recombinant Prion Protein with Organo-Mineral
Complexes as Assessed by FT-IR and CPMAS
13
C NMR Analysis
Fabio Russo
1
, Liliana Gianfreda
1
, Pellegrino Conte
2
and Maria A. Rao*
,1

1
Dipartimento di Scienze del Suolo, della Pianta, dellAmbiente e delle Produzioni Animali. Universit di Napoli
Federico II, via Universit 100, 80055 Portici (NA), Italy
2
Dipartimento di Ingegneria e Tecnologie Agro-Forestali. Universit degli Studi di Palermo, v.le delle Scienze 13,
edificio 4, 90128 Palermo, Italy
Abstract: Prion proteins are considered as the main agents for the Transmissible Spongiform Encephalopathies (TSE).
The misfolded form, PrP
Sc
, which is also indicated as the etiological agent for TSE, exhibits high resistance to degradation
in environmental processes. Soil contamination by prion proteins is a real environmental issue since contaminated soils
can become potential reservoir and diffuser for TSE infectivity. In this work, the interaction of prion protein with organo-
mineral complexes was studied by using a recombinant non pathogenic prion protein and a model soil system. This latter
was represented by a soil manganese mineral coated with polymerized catechol. FT-IR spectra showed amide I and II sig-
nals which revealed protein involvement in catechol polymers coating manganese oxide surface. CPMAS
13
C-NMR was
applied to follow the complexation of the protein to the soil-like system. All the signals were attributed to C-N in peptidic
bonds, alkyl chains and methyl groups. The NMR spectrum of the prion protein interacting directly with birnessite re-
vealed disappearance of signals due to the paramagnetic nature of manganese oxide or protein abiotic degradation, while
the presence of organic matter strongly reduced the disappearance of prion protein signals.
Keywords: Prion protein, TSE diseases, Soil Organo-Mineral Complexes, FT-IR, CPMAS
13
C NMR.
INTRODUCTION
Transmissible Spongiform Encephalopathies are fatal,
neurodegenerative diseases also known as prion diseases
including bovine spongiform encephalopathy, human
Creutzfeldt-Jakob disease, kuru, sheep scrapie, and chronic
wasting disease of deer, elk and moose [1]. PrP
Sc
is a mis-
folded isoform of the normal cellular prion protein (PrP
C
)
considered as the main, if not the sole, agent of TSE [2].
PrP
Sc
causes pathogenesis in the central nervous system with
the formation of amyloidal aggregates and a consequent
spongiosis in the brain of humans and animals. These dis-
eases, nowadays considered incurable, lead slowly but in-
exorably to death.
PrP
Sc
is protease resistant and exhibits a remarkable resis-
tance to degradation and inactivation by standard decontami-
nation procedures [3].
Dispersion of PrP
Sc
contaminated material in soil can oc-
cur from meat and bone meal storage plants, use of fertilizers
augmented with meat and bone, decomposition of animal
carcasses buried in soil, liquid and solid waste fragments
from abattoirs, and wastes from TSE-infected animals [4].
As firstly evidenced by Brown and Gajdusek [5], buried in-
fectious prion protein can persists in soil even for years. Sei-
del et al. [6] found that in soil, scrapie agent remained in an
infectious form after 29 months. Moreover, infected soil

*Address correspondence to this author at the Dipartimento di Scienze del
Suolo della Pianta, dellAmbiente e delle Produzioni Animali, Universit
degli Studi di Napoli Federico II, via Universit 100, Portici, Italy; Tel:
00390812529173; Fax: 00390812539186; E-mail: mariarao@unina.it
was supposed to preserve TSE capability to transmit the dis-
ease to healthy grazing animals after 16 years from the con-
tamination event [7].
Either the infective or the benign forms of prion proteins
are able to strongly bind to mineral [8-12] and organic soil
components [13-15]. PrP
Sc
can be displaced from environ-
mental compartments to soil, but this does not necessarily
decrease its infectivity [10,16]. Prion protein and other soil
exogenous proteins can also easily interact with organo-
mineral complexes that likely are in soil more abundant than
the mineral or organic components alone. Soil has been sug-
gested to act as a reservoir of TSE infectivity [17,18] and
even to enhance the transmissibility of prion disease by oral
uptake of soil complexed PrP
Sc
[16]. While biotic PrP
Sc
deg-
radation by some proteases can be reduced because of persis-
tent PrP
Sc
aggregates, its inactivation may arise by abiotic
oxidative reactions. In fact birnessite, a manganese oxide
common in soil, is able to degrade the PrP
Sc
in aqueous sus-
pensions [19]. The presence of organic matter coating reac-
tive soil mineral surfaces could hinder abiotic degradation
processes with different effects on prion stability.
Interaction of PrP
Sc
with organic matter can take place ei-
ther with forming or pre-formed humic substances (HS).
Interaction with already-formed HS could lead to superficial
protein sorption, while entrapment phenomenon could arise
if the protein is involved in the humification process [13]. In
general, immobilized proteins may result more stable than
free forms; for instance enzymes in humic complexes are
more resistant to thermal denaturation and proteolysis than
the respective free enzymes [20].
Interaction of a Recombinant Prion Protein The Open Magnetic Resonance Journal, 2010, Volume 3 85
In soil, prion proteins can interact with soil constituents
and remain immobilized in soil colloids. Since in soil envi-
ronment prion protein contamination can likely occur at the
surface layers, the infectious agent can result more easily
available for free-ranging animals promoting the disease
diffusion in the environment. It is, therefore, important to
understand how prion proteins interact with soil components
present in the surface layers and how the soil organic matter
and its transformation in humic substances could affect the
stability of prions, possibly protecting them from environ-
mental degradation.
The aim of the present work was to study through differ-
ent spectroscopic techniques, such as FT-IR and CPMAS
13
C-NMR, the complexation of a recombinant ovine prion
protein (recPrP) with soil organo-mineral complexes ob-
tained by oxidative polymerization of catechol mediated by
birnessite. The birnessite-catechol complexes resembling soil
organo-mineral complexes were prepared in the presence of
recPrP and by following two different sequences of addition
of recPrP, before and after catechol polymerization, in order
to obtain either entrapment or sorption, respectively.
Chemicals
Reagent grade catechol (Cat) (>99 % purity) and HPLC
grade solvents were purchased from Sigma Aldrich (Ger-
many). Birnessite (Bir) was synthesized according to
McKenzie [21] using KMnO
4
and HCl. X-ray spectra dif-
fraction analyses performed on the synthesized MnO
2
con-
firmed the birnessite poorly crystalline mineral structure
with characteristic peaks as reported in McKenzie [21]. Bir-
nessite has a point of zero charge of 1.81 [22], and pH-
dependent negative charge at typical soil pHs.
A purified recombinant ovine ARQ genetic variant prion
protein (recPrP) with a MM of 23 kDa (residues 23-234) was
prepared according to Rezaei et al. [23] and kindly furnished
by Virologie et Immunologie Molculaires lab of INRA
(Jouy-en-Josas, France). The protein with an isoelectric point
of 9.2 [23] is constituted by a well-folded C-terminal domain
(residues 125-234) and a flexible N-terminal arm (residues
23-124) [24].
Preparation of recPrP-Organo-Mineral Complexes
Organo-mineral-recPrP complexes used in this study
were prepared according to the methodology described by
Rao et al. [13]. Briefly, either 3 or 5 mM catechol (Cat3 and
Cat5, respectively), 0.5 mg ml
-1
recPrP and 5 mg ml
-1
birnes-
site in 0.1 M sodium acetate buffer at pH 5.5 were incubated
in different systems. Two ternary samples (catechol-
birnessite-recPrP) were produced: Cat-Bir-recPrP, where
Cat, Bir and recPrP were incubated for 4 h at 25 C, and Cat-
Bir+recPrP, where Cat and Bir were incubated for 2 h before
adding recPrP and incubating for further 2 h at 25 C. Binary
systems, Cat-Bir, Cat-recPrP and Bir-recPrP, and sample
with only Cat, recPrP or Bir were also produced and served
as controls.
All trials were incubated for a total of 4 h at 25 C. Su-
pernatants and insoluble precipitates were separated by cen-
trifugation (30 min at 10,000 rev min
-1
and 4 C). Residual
catechol and recPrP concentrations were evaluated by HPLC
analysis [13] with a Shimadzu instrument equipped with
UV-Vis variable-wavelength absorbance detector, set at 280
and 222 nm respectively.
The pellets were washed twice with 0.02 M NaCl, twice
with double-distilled water, freeze-dried and stored at 4 C
[13].
FT-IR Analyses
The Fourier transform infrared spectra of insoluble prod-
ucts were recorded by the Universal Attenuated Total Re-
flectance (UATR) method using a Perkin Elmer FT-IR spec-
trometer. Each spectrum represents a collection of 24 scans
recorded at a 4 cm
-1
resolution.
CPMAS
13
C-NMR Analyses
CPMAS
13
C NMR measurements were performed on a
Bruker Avance-II 400 spectrometer (Bruker Biospin, Milan,
Italy) operating at 100.6 MHz on carbon-13 and equipped
with a 4 mm standard bore solid state probe. Samples were
packed into 4 mm zirconia rotors with Kel-F caps and the
rotor spin rate was set at 13,000 1 Hz. In total, 4 k data
points (TD) were collected over an acquisition time (AQ) of
50 ms, a recycle delay (RD) of 2.0 s, and 30,000 scans (NS).
Contact time was 1 ms, while a
1
H RAMP sequence was
used to account for possible inhomogeneity of the Hart-
mannHahn condition at high rotor spin rates [25]. Precise
1
H 90 pulse calibration for CP acquisition was obtained as
reported in Conte and Piccolo [26]. Bruker Topspin
2.0
software was used to acquire all the spectra. Spectra elabora-
tion was conducted by Mestre-C software (Version 4.9.9.9,
Mestrelab Research, Santiago de Compostela, Spain). All the
FIDs (free induction decays) were transformed by applying
first a 4 k zero filling and then an exponential filter function
with a line broadening (LB) of 100 Hz. Fully automatic
baseline correction using a Bernstein algorithm was applied
for baseline corrections [27].
RESULTS
FT-IR Analyses
Infrared spectra were recorded for polymeric products of
catechol adsorbed on birnessite surface and their complexes
with recPrP added under different sequences (Fig. 1).

Fig. (1). FT-IR spectra of A. Bir; B. Bir-Cat3; C. Bir-Cat3+recPrP;
D. Bir-Cat3-recPrP.
86 The Open Magnetic Resonance Journal, 2010, Volume 3 Russo et al.
The most characteristic bands and their assignments re-
lated to samples obtained with 3 mM Cat as well as 5 mM
Cat (data not shown) are summarized in Table 1.
All samples showed a broad band at 3333 cm
-1
attribut-
able to OH groups bound through intermolecular H bonds
[28]. Spectral bands derived from vibrations of aromatic
carboxylates (R-COO
-
) and/or aromatic C=C structures
(1650-1620 cm
-1
) were also present. FT-IR spectra revealed
the presence of organic material attributed to the presence of
Cat polymerization products, (1621 and 1255 cm
-1
). Com-
plexes containing recPrP showed typical signals of amide I
and amide II (1645 and 1520 cm
-1
, respectively); a rein-
forcement of the weak signal at 1255 cm
-1
was attributed to
amide III. Similar FT-IR spectra were recorded for catechol
birnessitePrP complexes obtained at 5 mM catechol [13].
CPMAS
13
C NMR Analyses
RecPrP was analysed by CPMAS
13
C NMR (Fig. 2A).
The spectrum revealed a typical NMR signal pattern for pro-
teins in the solid state [29]. In fact, a signal attributable to
C=O groups was observed at 178 ppm, while signals at 161,
133, 120 ppm were assigned to aromatic moieties having
different substitution degrees [30]. All the signals comprised
between 90 and 0 ppm were attributed to C-N in peptidic
bonds (80 ppm), alkyl chains (47 ppm) and methyl groups
(34 ppm), respectively. The spectra of the birnessite with
prion protein or catechol (Bir-recPrP, Bir-Cat5 and Bir-Cat3,
respectively, Fig. 2B,C,D) appeared resolution-less, whereas
those of samples obtained by interaction of birnessite, phenol
and prion protein added before and after catechol polymeri-
zation process (Bir-Cat3+recPrP and Bir-Cat3-recPrP, re-
spectively, Fig. 2E,F) revealed the same signal pattern of
recPrP alone as in Fig. (2A), but with a larger signal width.
DISCUSSION
The recPrP was completely removed from the solution by
sorption or entrapment in catechol-birnessite soil-model sys-
tems confirming the high affinity of prions to soil organo-
mineral colloids. The presence in the insoluble catechol-
protein polymeric products of humic-like compounds and of
the protein was confirmed by FT-IR signals typical of humic
compounds and proteins (Table 1).

Fig. (2). CPMAS
13
C-NMR spectra of A. recPrP; B. Bir-recPrP; C.
Bir-Cat5; D. Bir-Cat3; E. Bir-Cat3+recPrP; F. Bir-Cat3-recPrP.
Table 1. Location of Relevant Indicator Bands in recPrP and Organo-Mineral Complexes and the Assignment to Functional
Groups
Wavenumber (cm
-1
) Sample Assignment
3333 Cat3-Bir
Cat3-Bir-recPrP
Cat5-Bir+recPrP
OH, bonded through intermolecular H bonds
1621 Cat3-Bir Aromatic C=C, H-bonded C=O or alkenes in conjugation with C=O
1645 recPrP
Cat3-Bir-recPrP
Cat3-Bir+recPrP
C=O stretching (amide I)
1520 recPrP
Cat3-Bir-recPrP
Cat5-Bir+recPrP
N-H bending (amide II)
1255 recPrP
Cat3-Bir
Cat3-Bir-recPrP
Cat5-Bir+recPrP
C-O stretching and aromatic C=C; amide III

Interaction of a Recombinant Prion Protein The Open Magnetic Resonance Journal, 2010, Volume 3 87
Catechol transformation by birnessite started to produce
soluble compounds as detected in the supernatants by UV-
Vis analyses. They were more abundant in the samples with
3 mM catechol than with 5 mM catechol [13]. When the pro-
tein was added before catechol polymerization (Cat-Bir-
recPrP) as well as when catechol was preventively polymer-
ized and partially adsorbed on birnessite surfaces (Cat-
Bir+recPrP) recPrP interacted with birnessite-catechol com-
plexes and was completely removed from solution, as dem-
onstrated by HPLC analysis [13]. Conversely in Bir-recPrP
sample, 33% of recPrP was detected as free in the super-
natant. The stability of recPrP in the humic-like complexes
was confirmed by the negative release of the protein after
extractions with several, even strong, extractants [13].
As reported in literature, a partial recPrP abiotic degrada-
tion mediated by MnO
2
could be not excluded [19]. How-
ever, in the presence of catechol the degradation of recPrP
should have been reduced because birnessite active surfaces
were covered by organic catechol polymers [31,32]. After
catechol polymer deposition, a reduced birnessite activity is
reasonable because the birnessite-mediated reaction is re-
ported to be chemically surface-controlled and occurring by
associative ligand substitution mechanism with a surface
complex formation on Mn-oxide sites [22]. According to this
mechanism, during the preparation of the complexes,
catechol produced large polymers located at the surface of
birnessite and capable of promoting the formation of large
pores among the enclosed birnessite particles [15].
As also reported in Rao et al. [13], UV-Vis spectra and
elemental analyses of the samples produced with 3 mM
catechol solution showed a larger soluble polymeric content
while samples produced with a 5 mM catechol solution had a
dominant insoluble catechol polymeric fraction. The weaker
bands of amide I and II observed in Cat3-Bir+recPrP than
those of Cat5-Bir+recPrP confirmed their different behav-
iour. In any case, the initial catechol concentration and the
sequence of the addition of the protein had no effect on pro-
tein immobilization that resulted always completely removed
from the solution.
Acquisition of solid state (SS) NMR spectra is strongly
affected by paramagnetism [33]. In fact, the presence of par-
amagnetic systems (PS) alters NMR signal shape and inten-
sity, thereby providing no signal at all when the amount of
paramagnetic species is larger than that of the NMR investi-
gated nucleus. It is reported, for example, that iron (III) in
soil samples prevents reliable
13
C NMR spectra acquisition
when the Fe/C mass ratio is 1 [25].
NMR signal disappearing is attributed to the interactions
between the magnetic field generated by the paramagnetic
centers and the applied magnetic field [34]. These interac-
tions fasten both longitudinal (T
1
) and transversal (T
2
) re-
laxation times so that NMR signals are both broadened and
lowered [25]. In the solid state, an additional problem is the
mismatching of the fundamental SS NMR condition,
T
CH
T
1
(H), which usually guarantees obtainment of quanti-
tative NMR spectra [25]. However, from a qualitative point
of view, when a spectrum is acquired, the relative intensities
of the various resonances do not reflect the real distribution
present in the samples since some functional groups may be
more affected by the presence of the paramagnetic centers
[35]. In fact, all the groups directly interacting with the par-
amagnetic ions relax faster than those placed far away from
PSs. As a consequence, the NMR signals of the quick relax-
ing nuclei disappear more rapidly than the resonances of the
remaining others.
Fig. (2A, B) reports the CPMAS
13
C NMR spectra of the
recombinant prion protein before (Fig. 2A) and after (Fig.
2B) absorption on birnessite (Bir). Due to the presence of the
paramagnetic manganese, all the signals of recPrP were
broadened. However, a broad signal around 130 ppm due to
aromatic structures and the resonance at 178 ppm due to car-
boxyls were still visible. Only the signals between 90 and 0
ppm completely disappeared (Fig. 2B). A possible explana-
tion for such behaviour can be related to the preferential in-
teractions of recPrP with the surface area of birnessite
through C-N and alkyl moieties.
The effect of natural organic matter on recPrP absorption
on birnessite was estimated by analyzing the complexes ob-
tained with catechol at the two different concentrations (3
and 5 mM, respectively) (see Materials and Methods). Fig.
(2C, D) shows that the humic-like organic substances pro-
duced by the incubation of catechol are adsorbed on birnes-
site. In fact, the signals of the incubated catechol disappeared
and only a very large band around 130 ppm became visible.
When the incubation of catechol-birnessite mixture was con-
ducted also in the presence of recPrP, added either after or
before catechol polymerization, the spectra in Figs. (2E and
2F) were obtained, respectively. Both spectra resembled that
in Fig. (2A), thereby indicating that only a humic-like medi-
ated interaction between recPrP and birnessite was possible.
CONCLUSIONS
The interaction of recPrP with catechol-birnessite com-
plexes occurred at all phenol concentrations used in the pre-
sent study. Moreover, the interactions took place regardless
of the sequence used to add the natural-organic-matter-like
(NOM-like) system to the mineral phase. In fact, recPrP ap-
peared to be bound to the birnessite through NOM-like me-
diated bindings which were observed by comparing the
CPMAS
13
C NMR spectra of different birnessite-recPrP
complexes. FT-IR analysis contributed to confirm the pres-
ence of recPrP in the insoluble polymeric products, already
at the lowest catechol concentration. Both FT-IR and NMR
studies were helpful to highlight the important role of humic-
like substances formed by abiotic catalysis from phenolic
compounds in adsorbing and/or entrapping recPrP, and the
prevalent involvement of alkyl moieties rather than aromatic
or carboxylic groups.
ACKNOWLEDGEMENTS
This work was founded by the European Union Project
QLK4-CT-2002-02493 TSE-Soil-Fate. The NMR experi-
ments were done at the Centro Grandi Apparecchiature
(CGA) - UniNetLab of Universit degli Studi di Palermo
(http://www.unipa.it/cga).
ABBREVIATIONS
recPrP = recombinant prion protein
Cat-Bir-recPrP = recPrP added before catechol
polymerization
88 The Open Magnetic Resonance Journal, 2010, Volume 3 Russo et al.
Cat-Bir+recPrP = recPrP added to catechol preventively
polymerized and partially adsorbed
on birnessite surfaces
CPMAS
13
C NMR = cross polarization magic angle spin-
ning carbon-13 nuclear magnetic
resonance
FT-IR = Fourier transform infrared spectros-
copy
PS = paramagnetic systems
SS NMR = solid state nuclear magnetic reso-
nance
T
CH
= cross polarization time
T
1
(H) = proton longitudinal relaxation time in
the rotating frame
T
1
= longitudinal relaxation time
T
2
= transverse relaxation time
UV-Vis = ultraviolet visible spectrophotometry
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Russo et al.; Licensee Bentham Open.


1874-7698/10 2010 Bentham Open
Open Access
CPMAS
13
C NMR Characterization of Leaves and Litters from the
Reafforestated Area of Mustigarufi in Sicily (Italy)
Pellegrino Conte*
,1
, Claudio De Pasquale
1
, Etelvino H. Novotny
2
, Gianluca Caponetto
1
,
Vito Armando Laudicina
1
, Maurizio Ciofalo
1
, Michele Panno
1
, Eristanna Palazzolo
1
,
Luigi Badalucco
1
and Giuseppe Alonzo
1

1
Dipartimento di Ingegneria e Tecnologie Agro-Forestali (ITAF), Universit degli Studi di Palermo, 90128 Palermo,
Italy
2
Embrapa Solos, Rua Jardim Botnico, 1024, CEP 22460-000, Rio de Janeiro, RJ, Brazil
Abstract: Reafforestation is generally based on the planting of exotic fast growing tree species suitable for adapting to
even harsh environments. Once the introduced plants ameliorate soil conditions, they can be progressively replaced by au-
tochthonous plant species. Reafforestation is applied worldwide. However, only few studies on the effect of reafforesta-
tion on lands from Mediterranean regions are available. This paper reports the characterization by cross polarization
13
C
NMR spectroscopy of fresh leaves and superficial litters from a reafforestated area in central Sicily (Italy). NMR assign-
ment is attempted. A differentiation among the molecular systems within leaves and litters is also done on the basis of
NMR assessment. Results showed that the main differences among the leaves of four forest trees (two eucalyptus spp.,
one cypress sp. and one pine sp.) occur in the distribution of the aromatic and alkyl carbons. In particular, the alkyl moie-
ties in the eucalyptus spp. leaves were attributed to branched structures belonging to the eucalyptus oil, whereas linear
fatty acids were more representetive in the NMR spectra of pine and cypress leaves. In addition, the aromatic carbons of
the conifer leaves were assigned not only to lignin- and tannin-like structures, but also to common olefin carbons in un-
saturated fatty acids and abietic acid-like systems. The spectra of the litters resembled, as expected, those of the leaves.
However, the presence of very large carbohydrate NMR signals suggested that degradation processes were still ongoing in
litters. A comparative evaluation of CPMAS
13
C NMR spectra was done by applying principal component analysis. This
paper confirmed the suitability of CPMAS
13
C NMR spectroscopy in evaluating the differences among natural bio-masses
which are the major nutrient sources for soil micro-organisms and the main input for humification processes.
Keywords: NMR, leaves, litters, reafforestation, degraded lands, soils.
INTRODUCTION
Intensive land use and management as well as inappro-
priate land practices have negative impacts on natural re-
sources such as waters, soils, atmosphere, plants and animals
due to nutrients decline, erosion and contamination [1, 2].
Therefore, land recovery and restoration are desirable efforts
for the improvement of the ecosystem health status and
sustainability [3]. Recovery and restoration are, however,
complex and long processes which imply many tasks includ-
ing environmental evaluations (such as pollution and/or ero-
sion extents), strategic policies for managing degraded eco-
systems, and technical accomplishments for rebuilding
physical and biological ecosystem conditions [3].
One of the most used practices for restoration of the natu-
ral ecosystems on abandoned lands (i.e. lands which ex-
hausted their natural potential for human survival) is the re-
afforestation [1, 4]. It consists in the planting of fast growing
exotic tree species suitable to adapt to even harsh conditions,

*Address correspondence to this author at the Dipartimento di Ingegneria e
Tecnologie Agro-Forestali (DITAF), Universit degli Studi di Palermo, v.le
delle Scienze 13, ed. 4, 90128, Palermo, Italy; Tel: 00390917028145; Fax:
0039091484035; E-mail: pellegrino.conte@unipa.it
thus providing early substrates for the microbial re-
colonization. Then, local species can be added by enrichment
planting to improve microclimate and soil conditions and to
create favorable circumstances for other indigenous species
invasion [5].
Reafforestation compensates for CO
2
emissions to the
atmosphere through accumulation and transformation of
natural organic matter into soils [2], promotes soil hydro-
logical properties due to its impact on soil texture [4], favors
mitigation of soil temperatures due to the vegetation cover
[4] and re-establishes nutrient cycles thereby affecting de-
velopment of soil microbial biomasses [6, 7]. Moreover,
reafforestation reduces soil erosion and may have a great
social impact since it increases economic potentials through
enhancement of working possibilities [8].
Land restoration through reafforestation is achieved
worldwide by establishing mainly eucalyptus and pine trees
[1]. In fact, such plantations take deep roots and grow vigor-
ously even in low fertility lands [4].
Only few papers up to now dealt with the effects of reaf-
forestation on Mediterranean degraded lands [7, 9, 10]. For
this reason, we have started the monitoring of the effects of
reafforestation in soils of semi-arid Mediterranean areas of
central Sicily (Italy) where desertification processes, related
90 The Open Magnetic Resonance Journal, 2010, Volume 3 Conte et al.
to intensive land uses, were ongoing. One of these areas is
the regional forest property located in Mustigarufi, nearby
Caltanissetta. Here eucalyptus, cypress and pine trees were
planted since the late fifties and the beginning of the sixties.
In the present paper, we have concentrated our attention
primarily on the molecular characterization of the fresh
leaves and the superficial layers of litters of Mustigarufi for-
est. This because, both of them represent the major nutrient
sources for soil microbial biomass and the main input for
humification processes, which are very important to restore
soil quality in reafforestated areas.
To reach our goal we applied carbon-13 solid state nu-
clear magnetic resonance spectroscopy with cross polariza-
tion and magic angle spinning (CPMAS
13
C NMR). CPMAS
13
C NMR spectroscopy provides both qualitative fingerprint-
ing of carbonaceous materials and quantitative measure-
ments on the relative content of the different molecular
moieties in very complex organic mixtures [11]. Therefore,
we report the assignment of the CPMAS
13
C NMR spectra of
fresh leaves and litter superficial layers from Mustigarufi
forest as well as a comparison among the molecular charac-
teristics of these materials. This study represents an unavoid-
able initial step for the integrated study of the C turnover
within a reafforested semi-arid Mediterranean area.
EXPERIMENTAL
Leaf and Litter Samples
Eucalyptus camaldulensis Dehnh. (EC), Eucalyptus occiden-
talis Endl. (EO), Cupressus sempervirens L. (CI) and Pinus
halepensis Mill. (PI) are tree species growing in Mustigarufi
reafforested area which is located nearby San Cataldo (Cal-
tanissetta, Sicily, Italy, 3733N, 1355E) on a hill placed
at 470 m a.s.l.. In order to perform the sampling of fresh
leaves and superficial litters, four distinct 20 x 20 m homo-
geneous squares, each including only one tree species, were
selected. Composite samples of fresh leaves of each tree spe-
cies were collected at random from three distinct canopies,
whereas composite samples from superficial (0-3 cm) litters
consisted of six sub-samples exploring the 20 x 20 m
squares. It was not possible to collect the litter under Cu-
pressus sempervirens L. since it appeared both heterogene-
ous and quantitatively insignificant. Leaves and litters,
without any pre-treatment, were dried at 70C in a vent-oven
for 72 hours. Then, they were powdered in an ultra centrifu-
gal rotor ZM200 Retsch

mill equipped with a 1 mm sieve
in order to obtain solid samples for the NMR analyses.
CPMAS
13
C NMR Spectroscopy
CPMAS
13
C NMR measurements were performed on a
Bruker Avance-II 400 spectrometer (Bruker Biospin, Milan,
Italy) operating at 100.6 MHz on carbon-13 and equipped
with a 4 mm standard bore solid state probe. Samples were
packed into 4 mm zirconia rotors with Kel-F caps and the
rotor spin rate was set at 13000 2 Hz. A spectral width of
29761.90 Hz centered at 10061.78 Hz, an optimum contact
time of 1 ms chosen after evaluation of variable contact time
experiments [12], a recycle delay of 2 s, 2 k data points over
an acquisition time of 35 ms and a RAMP sequence, to ac-
count for inhomogeneities of the Hartmann-Hahn condition
at high rotor spin rates [11], were used. 700 scans were ac-
cumulated to obtain all the spectra. The
1
H 90 pulse was
calibrated on each leaf and litter sample with the pulse se-
quence described in [13] at an attenuation level of -2.4 dB.
1
H 90 pulse length varied within the interval 3.60-3.85 s
depending on the sample under analysis. Spectra acquisition
was achieved with Bruker Topspin
2.0. Data elaboration

was done with MestRe-C software (Version 4.9.9.9, Mestre-
lab Research, Santiago de Compostela, Spain). The free in-
duction decays (FIDs) were transformed by applying first a 2
k zero filling, then a line broadening of 50 Hz and finally an
automatic baseline correction with a 3rd order polynomial
and Bernstein algorithm [14]. All the spectra were divided in
the following regions whose assignment is fully discussed
later: 0-45 (alkyl C), 45-60 (O/N alkyl C), 60-90 (O alkyls in
carbohydrates), 90-120 (acetal and lignin C), 120-160 (aro-
matic C) and 160-180 (-COOH groups) ppm. The absolute
areas (A
i
) of each interval was measured and referred to the
total absolute area of each spectrum (A
T
) as obtained by in-
tegrating the -50 230 ppm interval. The -50 230 ppm
region was accounted for to consider the effect of the spec-
tral noise on the quantitative evaluation of the NMR spectra.
Principal Component Analysis
Principal Component Analysis (PCA) was carried out us-
ing the CPMAS
13
C NMR spectra obtained, after area nor-
malization and mean-centering of the data. To improve the
interpretability of the loadings, a Varimax rotation was per-
formed. The goal of this strategy was to obtain a clear pat-
tern of loadings, i.e., factors marked by high loadings for
some variables and low loadings for others. The model vali-
dation was carried out using two different methods: full
cross-validation, and segmented cross-validation with two
samples per segment picked at random. The difference in the
variance between the calibration and validation models was
less than 5%. This analysis was performed using The Un-
scrambler software (CAMO Software AS, Oslo, Norway).
Qualitative Interpretation of the Leaf NMR Spectra
Fig. (1) reports the CPMAS
13
C NMR spectra of the
leaves sampled for the present study. Attribution of spectral
regions is also indicated. According to literature [11, 16-19],
six different intervals were recognized. The first one, be-
tween 0 and 45 ppm, was attributed to alkyl systems. The
most important signals in this interval were located at 26, 30
and 32 ppm in the spectra of Cupressus sempervirens L. (CI,
Fig. 1A) and Pinus halepensis Mill. (PI, Fig. 1B) and at 17,
26, 30, 32, 39 and 42 ppm in the spectra of Eucalyptus
camaldulensis Dehnh. (EC, Fig. 1C) and Eucalyptus occi-
dentalis Endl. (EO, Fig. 1D).
The resonance at 17 ppm was assigned to methyl groups
which terminate alkyl chains [16]. Among the other remain-
ing alkyl signals (26, 30, 32, 39 and 42 ppm), those at 26, 30
and 32 ppm, visible in all the leaf spectra (Fig. 1), can be
attributed to linear methylene (-CH
2
-) chains [16, 17] be-
longing to lipids, cutin-like structures and other aliphatic
bio-moieties [20]. The last two, at 39 and 42 ppm can be
assigned to secondary methyne carbons (-CH-, signal at 39
ppm) and to fully substituted quaternary carbons (CR
4
, signal
at 42 ppm) [16, 19]. It is likely that the signals at 39 and 42
ppm can be generated by carbons in chlorophyll-like struc-
NMR Characterization of Biomasses from a Reafforestated Italian Area The Open Magnetic Resonance Journal, 2010, Volume 3 91
tures or in molecules belonging to eucalyptus-oil (a complex
mixture of terpenoids) that is usually present into eucalyptus
trees [21].
The second spectral interval between 45 and 60 ppm is
traditionally attributed to nitrogenated and oxygenated alkyl
systems (Fig. 1). Three signals positioned at 48, 53 and 56
ppm were evidenced in the spectra of Fig. (1). That at 48
ppm can be assigned to N-alkyl carbons in amino acids [18,
22]. The shoulder at 53 ppm in Figs. (1C and 1D) and the
peak at 56 ppm in Figs. (1A to 1D), can be both attributed to
O-alkyl groups such as methoxyls in lignin-like structures
(56 ppm) and CH
2
O systems into branched molecules as
those in the eucalyptus-oil [18].
The region comprised in the chemical shift interval 60-90
ppm (Fig. 1) is indicative of carbohydrates with the largest
contribution due to celluloses and hemicelluloses [18]. In
particular, the resonances at 63 and 65 ppm were due to car-
bon 6 in amorphous and crystalline celluloses, respectively
[23], while the intensity at 72 ppm was assigned to the car-
bons in the positions 2, 3 and 5 regardless of the cellulose
nature (either crystalline or not) [24]. Carbon 4 appeared
between 80 and 90 ppm.
Here, it generated a shoulder at around 83 ppm due to
amorphous cellulose, hemicellulose and cellulose oligomers
[25] and a signal at 88 ppm that was assigned to the ordered
forms of celluloses on fibril surfaces and to in core para-
crystalline celluloses, whose nature is still uncertain [26].
Among all the spectra (Fig. 1), the only one where the sig-
nals at 63/65 and 83/88 ppm were clearly identifiable was
generated by the pine leaves (Fig. 1B), thereby suggesting
that they may contain a larger variety of cellulose forms.
Signals of carbohydrates are also observed in the range
90-120 ppm where the large resonance at 105 ppm was at-
tributed to C1 of cellobiose units into cellulose I, and that at
around 100 ppm was assigned to the acetal carbons in the
xylane systems of hemicelluloses [26]. The 90-120 ppm in-
terval contains also other signals such as a weak one due to
common olefin carbons at 116 ppm in the spectra of CI (Fig.
1A) and PI (Fig. 1B), and a resonance at 109 ppm associable
to acetal C in cellulose II.
The spectral region, that traditionally is assigned to aro-
matic systems, is included between 120 and 160 ppm [16].
Three main peaks at 130, 144 and 154 ppm were revealed in
the spectra of the leaves from CI (Fig. 1A) and PI (Fig. 1B),
whereas only a signal at 137 ppm and a large resonance at
144 ppm appeared in the spectra of EC (Fig. 1C) and EO
(Fig. 1D). All these signals are usually attributed to lignin
systems [17]. In particular, p-hydroxyphenol derivative
structures are assumed to give a signal at around 130 ppm,
whereas O-aryl carbons from guaiacyl- and syringyl-units
may give resonances at 137, 144 and 154 ppm [17]. Based
on this simple attribution, we can conclude that the differ-
ences between the leaves from the two coniferous trees and
the two eucalyptus plants were due to a different concentra-
tion of p-hydroxyphenol-, guaiacyl- and syringyl- structures.
However, cell composition of the needles from pinus and
cypress has been already well studied and it has been clari-
fied that they contain large concentrations of acid resins
made by long chain unsaturated acids and abietic acid de-
rivatives [27, 28]. According to web data bases (such as
http://riodb01.ibase.aist.go.jp/sdbs/) and to literature data
[29], we attributed the resonance at 130 ppm in the spectra of
CI (Fig. 1A) and PI (Fig. 1B) either to cis-mono-unsaturated
fatty acids or to abietic acid-like structures (i.e. sp
2
carbons).
The signal at 144 ppm can be, consequently, also assigned to
the C13 in dehydro-phenanthrene systems [29]. According to
our interpretation, the differences in the 120-160 ppm inter-
val in the leaf spectra of the coniferous plants (Fig. 1A and
1B) and of the two eucalyptus trees (Fig. 1C and 1D) cannot
be due only to a different distribution of p-hydroxyphenol-,
guaiacyl- and syringyl- structures, but also to the presence of
acid resins which are less abundant in the leaf samples from
EC and EO.
The last spectral region between 160 and 180 ppm was
attributed to COOH and amide groups.
Qualitative Interpretation of the Litter NMR Spectra
Signal attribution of the litter spectra (Fig. 2) is similar to
that of the leaves. It has been already described in the para-

Fig. (1). CPMAS
13
C NMR spectra of the leaves of Cupressus
sempervirens L. (A), Pinus halepensis Mill. (B), Eucaliptus
camaldulensis Dehnh. (C) and Eucaliptus occidentalis Endl. (D).
Attribution of the spectral intervals is also reported.

graph above. As a general remark, however, it must be no-
ticed that all the litter NMR spectra are dominated by the
signals of carbohydrates. According to Lemma et al. [18]
and Hopkins et al. [30], this feature reveals that the litters are
still in the early stages of decomposition. The comparison of
Figs. (1 and 2) shows some dissimilarities between fresh
leaves and leaf litters. Signal at 65 ppm (which appears as a
shoulder of the signal at 72 ppm in the leaf spectra) and a
different distribution of the aromatic (120-160 ppm) carbons
can be observed. The signal at 65 ppm was assigned to C6 in
crystalline cellulose (see above). The higher resolution of
such signal can be explained considering that the first steps
of the litter decomposition mechanisms involve the degrada-
tion of the amorphous celluloses [18]. As a consequence, a
sharpening of the signals in the O-alkyl spectral region,
which implies a better separation between the resonances at
72 and 65 ppm, is achieved. This hypothesis is further sup-
ported taking into account the disappearance of the signal at
109 ppm (in EO and EC litters) assigned to acetal carbons in
cellulose II. Litter decomposition is also related to a varia-
tion of the nature of the aromatic carbons due to degradation
of tannin- and lignin-like components and to a general reso-
lution-loss of the signals ranging in the region of the alkyl
moieties. Transformations of the aromatic structures turned
in the coalescence of the signals at 144 and 154 ppm which
was observed in the spectrum of the pinus litter (Fig. 2A)
[18]. The same signals (at 144 and 154 ppm) were, con-
versely, observed in the spectrum of EC litter (Fig. 2B).
They were not present in the spectrum of the EC leaves (Fig.
1C). The presence of these two signals can be attributed to
lignin-residues which were co-sampled with the leaf litter.
Statistical Comparison of NMR Spectra
In order to evaluate the capability of the CPMAS
13
C
NMR spectroscopy in revealing differences among leaf and
litter samples, the NMR spectra were used as input data for
principal component analysis (PCA). PCA is already known
to be a powerful tool for the recognition of similarities and
dissimilarities in NMR spectra of very complex systems such
as in foods [31-36], natural organic matter [25, 37-40], living
organisms [41], and environmental compartments [42-45].
The basic idea of PCA is to reduce the number of variables
into just few and to seek linear combinations of those vari-
ables explaining most of the variability [15].
In the present study, PCA reduced the number of vari-
ables to only 3 (PC1, PC2 and PC3) which accounted for
83% of the total variance (Fig. 3). Both PC1 and PC3 indi-
cate decomposition of Eucaliptus leaves. The sole PC1,
which accounted for the 35% of the variance, revealed that
Eucaliptus leaves had the largest scores as compared to the
Eucaliptus litters (Fig. 3A). Moreover, this component was
also able to differentiate among tree species due to the score
values which varied as EO>ECPI>CI.

Fig. (3). Scores of rotated (varimax) PCA from CPMAS
13
C NMR
spectra. CI=Cupressus sempervirens L.; PI=Pinus halepensis Mill.;
EO=Eucaliptus occidentalis Endl.; EC=Eucaliptus camaldulensis
Dehnh. A. PC1 vs PC2 plot; B. PC1 vs PC3 plot.

PC1 was characterised by high positive loadings in the
region of methyl groups (< 30 ppm), with clear peaks at

Fig. (2). CPMAS
13
C NMR spectra of the litters from Pinus
halepensis Mill. (A), Eucaliptus camaldulensis Dehnh. (B) and
Eucaliptus occidentalis Endl. (C).

16.8, 20.7, 24.0, 28.3 ppm and shoulders at around 11.9 and
18.4 ppm (Fig. 4). According to Fig. (3A), methyl systems
are more abundant in EO leaves (higher scores) which, in
turn, are subjected to an easier decomposition than the leaves
from the other tree species. PC1 also showed positive load-
ings (Fig. 4) in the region of methyne and quaternary groups
(39.5 and 41.8 ppm, respectively). Chlorophyll-like and oil-
like structures (see above) appeared to resonate at those
chemical shift values, thereby confirming that such struc-
tures are peculiar in Eucaliptus trees.

Fig. (4). Loadings of first rotated (varimax) Principal Component
from PCA of CPMAS
13
C NMR spectra.

The negative loadings for the signals at 32.8 (weak peak,
crystalline poly-methylene) and at 35.9 ppm are an indica-
tion that during the initial stage of decomposition a relative
accumulation of crystalline forms of poly-methylene may
occur.
The chemical shift interval comprising the signals at
54.7, 52.4, 49.6, and 47.5 ppm (Fig. 4) and the region 154.7
and 152.4 ppm revealed opposite loading signs. In fact, the
former region, generally attributable either to O-alkyl or N-
alkyl carbons, showed positive loadings, whereas the second
one, attributed to O-aryl groups, was negative (Fig. 4). Due
to this inverse relationship, it can be concluded that the main
organic systems which are subjected to decomposition are N-
alkyls in peptides.
In addition, Fig. (4) reports negative loadings for the
peaks due to crystalline cellulose (from 106 to 70.4 ppm)
and positive loadings for the peak of amorphous cellulose
(63.7 ppm), thereby revealing an accumulation of the crystal-
line cellulose, while the amorphous one is decomposed.
Finally, the aryl region showed negative loadings, proba-
bly due to a relative accumulation of lignin during the de-
composition.
PC3 provide additional information concerning the al-
terations of Eucalyptus leaves (Fig. 3B). In fact, Fig. (5)
shows positive loadings from the chemical groups which are
more abundant in Eucalyptus leaves and that sharply de-
crease in litter samples (i.e. 70 and 32 ppm). Conversely, the
negative signals at 104, 75 and 21 ppm (Fig. 5) belong to the
groups which accumulate in the litter.
PC2 accounted for 29% of the total variance (Fig. 3A). It
is characterized by positive loadings in the region 60-90 ppm
(data not reported). Its scores clearly reveal decomposition of
amorphous cellulose in PI-leaves while the crystalline one is
accumulated.

Fig. (5). Loadings of third rotated (varimax) Principal Component
from PCA of CPMAS
13
C NMR spectra.

CONCLUSIONS
The molecular characterization by solid state
13
C NMR
spectroscopy of leaves and litters from a reafforestated area
in Sicily (Italy) is reported. Leaves from two different euca-
lyptus plants and two conifers (pine and cypress) appeared
significantly discriminated in the alkyl and aromatic NMR
regions. In fact, the eucalyptus leaves were the richest in
alkyl systems, whereas the pine and cypress leaves resulted
to have the largest content of aromatic carbon. The aromatic
C region in the leaves of the conifers was assigned not only
to lignin- and tannin-like structures as reported in literature
[17, 20], but also to common olefin carbons in unsaturated
fatty acids (signal at 130 ppm) and abietic acid-like systems.
This suggested that the differentiation between the leaves of
the eucalyptus trees and the two conifers cannot be based
only on p-hydroxyphenol, guaiacyl- and syringyl- deriva-
tives, but also on the presence of acid resins which are well
known to be very abundant in conifer leaves.
The spectra of the leaf litters were dominated by the car-
bohydrate signals, thereby revealing that first stages of de-
composition were ongoing [30].
Principal component analysis revealed that the initial
stages of leaves decomposition leads to the preferential de-
composition of methyl, methyne and quaternary C, N-alkyl
and amorphous cellulose. On the other hand, this process
leads to a relative accumulation of lignin and crystalline cel-
lulose and crystalline poly-methylene.
CPMAS
13
C NMR spectroscopy turned out to be a very
powerful technique for the molecular characterization of
leaves and litters which, in turn, are very important for the
development of microbial communities in reafforestated
soils.

ACKNOWLEDGEMENTS
The NMR experiments were done at the Centro Grandi
Apparecchiature (CGA) - UniNetLab of Universit degli
Studi di Palermo (http://www.unipa.it/cga)
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Received: June 15, 2009 Revised: December 04, 2009 Accepted: December 07, 2009

Conte et al.; Licensee Bentham Open.



1874-7698/10 2010 Bentham Open
Open Access
13
C-NMR Chemical Shift Databases as a Quick Tool to Evaluate Structural
Models of Humic Substances
Christian Nyrop Albers*
,a,b
and Poul Erik Hansen*
,a

a
Department Science, Systems and Models, Roskilde University, Universitetsvej 1, DK-4000, Roskilde, Denmark
b
Department Geochemistry, Geological Survey of Denmark & Greenland, . Voldgade 10, DK-1350, Copenhagen,
Denmark
Abstract: Models for humic and fulvic acids are discussed based on
13
C liquid state NMR spectra combined with results
from elemental analysis and titration studies. The analysis of NMR spectra is based on a full reconstruction of the NMR
spectrum done with help of
13
C-NMR data bases by adding up chemical shifts of all substructures from the proposed
models. A full reconstruction makes sure that all carbons are accounted for and enables on the negative side to discuss
structural elements identified from recorded spectra of humic substances that cannot be observed in the simulated spec-
trum. On the positive side missing structural elements in the models can be suggested. A number of proposed structures
for humic and fulvic acids are discussed based on the above analysis.
Keywords: Humic acid, fulvic acid,
13
C NMR, spectral reconstruction.
INTRODUCTION
Humic substances (HS) are one of the most abundant or-
ganic materials on earth. They represent 30-75% of soil or-
ganic matter (SOM) [1, 2] and the majority of dissolved or-
ganic matter (DOM) in both fresh and salt waters. Tradition-
ally, HS from SOM has been subdivided into three fractions;
Fulvic acids (FA) soluble at all pHs, Humic acids (HA)
soluble at basic pHs and humin, not soluble in aqueous solu-
tions. In DOM, only FA and HA, and often mainly FA, rep-
resents HS [3]. Both HA and FA play a major role in binding
and, hence, fate of both organic compounds, including pol-
lutants like pesticides and polyaromatic hydrocarbons [4-7],
and inorganic materials, especially cations [8]. The capacity
for binding organic and inorganic pollutants as well as many
other properties of HS is determined by the presence of vari-
ous structural elements. Among those, aromatic structures
have been shown to be important for binding of aromatic
pollutants [5, 6] while carboxylic acid groups are important
for the complex binding of cations [8]. The molecular struc-
ture of HS is very variable and complex, and despite many
years of research, no agreement for a common structure has
been reached. There seems to be a general agreement that no
single structure can be found. However, during the years
very different representative models for HA and FA have
been proposed (see e.g. Figs. (2 & 3) and references therein).
These models have included very different structural
subunits as well as similar structural elements in very differ-
ent proportions. This variety of models and substructures can
be highly confusing if one seeks inspiration in the literature
e.g. to understand and predict interactions between a HS-
fraction and some pollutant as well as understanding other

*Address correspondence to these authors at the Department Science,
Systems and Models, Roskilde University, Universitetsvej 1, DK-4000,
Roskilde, Denmark; Tel: +45 46742432; Fax: +45 46743011;
E-mails: calbers@ruc.dk, poulerik@ruc.dk
properties of HS. One possibility to explain the variety of
structural models could be that they represent different envi-
ronmental compartments and hence different parent material.
Principal component analysis (PCA) and similar techniques
have proven quite useful in distinguishing between origin
based on measured functional groups and other parameters
[9, 10], but in these studies it was found that while FA and
HA fractions can easily be separated based on structural
characteristics, the same fraction obtained from different
soils are not so different. Soil and lignite HAs were clearly
separated, though, so the origin does have an effect as well.
The purpose of the present paper is to suggest ways of char-
acterizing different fractions of humic substances and humic
fractions from different environments using liquid state
NMR and
13
C NMR databases and to verify the validity of
the previously proposed models by such method.
In characterizing the complex humic substances it is ob-
viously advantageous to use as many descriptors as possible.
Some of these descriptors may be obtained using NMR. Both
1
H- and
13
C-NMR have been used, the latter also in the solid
state. Obvious 2D techniques are HETCOR, HMQC or
HSQC [11-14]. The result is a correlation between
1
H and
13
C chemical shifts. A plot of these parameters shows that
the
1
H and
13
C chemical shifts are largely proportional as has
been found in general [15], but in some cases such spectra
have proved useful, e.g. in identifying substructures of the
aromatic parts of HA and FA [11]. HMBC spectra have also
been included. The extra information of such spectra is to
some extent counteracted by the many extra resonances and
increased overlap (see e.g. Ref. [16]).
It is essential to distinguish between liquid and solid state
spectra as different rules about intensities are found. In the
present paper we concentrate on 1D
13
C liquid state spectra,
which have been used extensively to identify the presence of
functional groups such as ketones, carboxylic acids, amides,
oxygen substituted aromatic carbons, methoxy groups and
Evaluation of Structures of Humic Substances The Open Magnetic Resonance Journal, 2010, Volume 3 97
aliphatic side chains as well as small molecular fragments
such as carbohydrates and amino acids. Since
13
C-NMR is a
chemical shift based method, even the carbon skeleton itself
will give rise to widely different chemical shifts (Fig. 1). In a
broad sense the number of terminal carbons in aliphatic
chains, versus the central carbons and finally more branched
situations can be estimated from the shape of the aliphatic
signal. Other elements such as oxygen, nitrogen and sulphur
are detected by means of their substituent effects. Except for
HAs derived from lignite, very little sulphur is present so
this is really not an issue. For single oxygens the substituent
effects are so that both aliphatic and aromatic substituents
can easily be detected, with e.g. mono-phenolic structural
elements typically appearing around 160 ppm. For di- and
tri-phenolic elements, the picture is less clear, and some or
all of the O-substituted C-atoms will give rise to signals at
much lower frequency (Fig. 1e).
A structural model of HS may be constructed based on
identified structural elements, carboxylic acids, ketones aro-
matic carbons etc. A more holistic approach is to identify
structural elements, gather a possible model and reconstruct
the entire NMR spectrum of the proposed model [10, 13].
This has been demonstrated based on data using chemical
software like e.g. ChemDraw [13] or based on suggested
structures and data from data bases [10]. 2D NMR data bases
have also been constructed to identify structural elements
and with the intent to reconstruct spectra of complex mix-
tures like humic substances [16]. The entire 2D spectrum is,
however, not yet possible to match, and the easy access to
1D NMR data bases and the ability to find suitable data for
complex structures [17] have prompted this study of a series
of suggested models of HA and FA.
Liquid State
13
C-NMR
HA and FA were purified from clayey agricultural soil
(Orthic Luvisol, sampled from the A-horizon), sandy agri-
cultural soil (Humic Podzol, sampled from the B
h
-horizon),
clayey grassland soil (Luvisol, sampled from the A-horizon)
and sandy coniferous forest soil (aeolian sand in the begin-
ning of a podzolization, sampled from organic H-horizon
and B
h
-horizon), largely according to the IHSS standard pro-
cedure with slight modifications [10]. Briefly, the soil was
air-dried and then extracted first with 0.1M HCl followed by
extraction with 0.1M NaOH under a N
2
-atmposphere. HA
was precipitated at pH=1, re-dissolved in KOH + KCl under
a N
2
-atmosphere, re-precipitated at pH=1 and inorganic im-
purities were then removed with HCl/HF followed by dialy-
sis. Aldrich HA was purchased from Sigma-Aldrich (Stein-
heim, Germany) and purified to remove the large content of
inorganic impurities and FA as described previously [10].
Liquid state
13
C-NMR of humic fractions was performed
as follows: ~80 mg HS was dissolved in 680 l H
2
O:D
2
O
(4:1) and pH was adjusted with 10 M NaOH to pH=7 (FA)
or pH=11 (HA). Spectra were recorded on a Varian 600
Inova Spectrometer (Varian, Palo Alto, California), working
at 150 MHz on
13
C, using a 5 mm BB-probe. Spectral width
was set to 40000 Hz, and 50000-100000 transients were re-
corded with gated decoupling to suppress Nuclear Overhau-
ser Effects with: Delay between experiments = 1000 ms,
pulse width = 6 s (corresponding to a flip angle of 52
o
),
acquisition time = 438 ms. Spectra were also recorded with a
delay of 2000 ms between experiments but this gave no dif-
ferences in intensities. A line broadening (LB) of 50 Hz was
applied to all spectra and 3-trimethylsilyl propionate (TSP)
was used as an external reference. One spectrum of freshwa-
ter FA and marine HA was taken from the literature in order
to compare across a wider range of environmental compart-
ments.
Simulated Spectra
The spectral data base Modgraph NMRPredict (Mestre-
Lab Research) [17] was used to predict chemical shifts. The
software predicts chemical shifts with protonated COOH and
phenolic OH groups, while spectra of FA fractions were re-
corded at pH=7 where COOH groups are deprotonated, and
HA fractions were recorded at pH=11 where most phenolic
OH groups would also be deprotonated. In order to compare
recorded and simulated spectra, the predicted COOH chemi-
cal shifts were changed by adding 9 ppm to the values ob-
tained for the Aromatic COOH groups and 6 ppm to those
calculated for the Alkyl COOH systems. 9 and 6 ppm are

Fig. (1).
13
C chemical shifts of model structures.
a) b)
c) d)
e) f)

98 The Open Magnetic Resonance Journal, 2010, Volume 3 Albers and Hansen
reported to be the average chemical shift changes occurring
when deprotonation of carboxylic acids arises following the
reactions: Aromatic COOH (Ar-COOH) Aromatic COO-
(Ar-COO- ) and Alkyl-COOH Alkyl-COO-, respectively
[18]. Chemical shifts of phenols are also affected by the de-
protonation that occurs at pH=11, where the HA fractions
were dissolved. The number of phenolic structures for which
such a change in chemical shift has been reported, is scarce
and more important, very different changes in chemical
shifts have been observed, depending on numbers and types
of substituents on the ring [18-20]. Therefore no attempts
were made to correct this. For the predicted spectra of HA,
this gives a slight error around 160-170 ppm, while for the
FA fractions (analyzed at pH=7) this was not of concern.
Spectra were simulated on the assumption of Lorentz-formed
peaks with a line broadening (LB) of 400 Hz at half peak-
height.
Models of HS
A large number of model structures have been suggested
over the years. For the present study, we have chosen 11
structures, which present a wide range of the most recent
models within different humic fractions and deriving from
several environmental compartments (Figs. 2 & 3). The
models were chosen on the basis that they have been cited
often and/or are the newest representatives for their class of
HS. It is obvious that no single structure as such can be con-
structed for neither HS in general nor even for a specific
fraction of HS from a specific environmental compartment.
Most if not all of the authors who have proposed the struc-
tural models in Figs. (2 & 3), have emphasized that their
model is not the final solution to humic structures, but that
the structures should rather be considered as assemblies of
structural elements. Despite these reservations, the authors
have presented such very different structures, that they obvi-
ously cannot all be representative of some average struc-
ture of HAs or FAs.
13
C-NMR Spectra
Numerous
13
C-NMR spectra have been published during
the years. Spectra have been recorded with the HS either in
aqueous solutions or in the solid state. The intensities are
clearly different in the liquid and solid state spectra as
pointed out by Conte et al. [28] and as seen from Fig. (4).

Fig. (2). Structural models for humic acids (HAs), taken from the literature. a) Not further defined HA (Schulten & Schnitzer, 1993) [21]. b)
Not further defined HA (Stevenson, 1994) [1]. c) Agricultural soil HA (Albers et al., 2008) [10]. d) Agricultural soil HA. One of five sub-
structures (Diallo et al., 2003) [13] e) Terrestrial HA from commercial Acros Organics Humic Substance (Sein et al., 1999) [22]. f) Marine
HA (Harvey et al., 1983) [23].

30

35

40

CH3
N
OH
C H3
CH3 OH
O
O
OH
OH
O H O
O
CH3
OH
OH
O
OH
O
O
OH O
C H3
OH
O
O
OH O
OH
O H O
C H3
O
OH
O H O
N
CH3
O
OH
O
O
OH
N
CH3
O
C H3
O
OH
O
OH
O
OH
C H3 O H
OH
OH
O
O H
O
OH
OH
O
O
C H3
O
C H3
O
OH OH
OH
O
O H O
CH3
O H O
OH
O
O
OH
O H O
OH
O
OH
O
CH3
N
H
CH3
OH
O
OH
O O H
O
O H
N
H
C H3
O
O H
OH
C H3
O H
O H
O
OH
O
O H O
O
CH3
CH3
a)
O
O
O OH
OH
O
N
O
N H CH
3
N H O
OH
O
O H
O
O
O
O
CH
3
OH
O
O H
O
OH
O O H
O OH
OH
O H
O
O
O H
OH
O H
OH
O
OH
OH
O
OH
O
b)
O
O
O
O H
NH
OH
O H
O
OH O H
OH
OH
O
N
H
O
O
OH
O OH
O
O H
O
OH
N H
C H
3
O
OH
NH
O O H
CH
3
CH
3
C H
3
O H
O H
O
NH
O
O H
C H
3
CH
3
c)
C H
3
OH
O
OH
O
C H
3
O
OH
OH O
O H
O
OH
O O OH
O H
O
O
C H
3
O
f)
O H
O
O
NH
2
O
O
O H OH
O
O H O OH
OH
O
O H
N H
2
OH
OH e)
d)
O H
O
O
CH
3
O
O
O H
O
OH
O
O H
O
O H
O
OH
OH
OH
O
S
C H
3
OH
O
OH
O O
OH

Fig. (3). Structural models for fulvic acids (FAs) and whole soil HS from literature. a) Soil FA (Shin & Moon, 1996) [24]. b) Four structural
units representing Suwannee River FA (Leenheer & Rostad, 2004) [25]. c) Terrestrial FA from commercial Acros Organics Humic substance
(Alvarez-Puebla et al., 2006) [26]. d) Agricultural soil FA (Albers et al., 2008) [10]. e) Assembly of the partial structures included in a con-
ceptual model for whole soil SOM (Kleber et al., 2007) [27].
This could be a serious problem in an attempt to reconstruct
spectra. Recent experiments have shown that a lignite humic
acid could be separated into fractions with varying sizes and
aromatic and aliphatic contents [29]. This is explained by the
recently growing view that humic substances are su-
pramolecular assemblies of compounds having relatively low
molecular weights [30, 31]. This again could lead to loss of
signal in the aliphatic region and therefore underestimation
of the aliphatic region (0-45 ppm) if parts of the aliphatic
chains are in a hydrophobic core of very large micelles or
similar macro ensembles. However, as seen from our previ-
ous experiments combining NMR, elemental analysis and
titration studies a good balance was actually found between
the different types of data [10]. This can be elaborated as
follows for a typical agricultural field humic acid (SlA HA).
From elemental analysis the formula was determined as
C
65
H
65
O
32
N
5
[10]. This leads to 36 degrees of unsaturation
(DoU). Eight carboxylic acid groups could be identified ei-
ther from integration of liquid state
13
C-NMR or a little less
(5-6) from titration studies and in addition six carbohydrate
carbons and four amino acid C carbons could be deter-
mined from the NMR experiments [10]. Subtracting the av-
erage of the two estimates of carboxylic acids as well as car-
bohydrate and amino acid carbons, the identified units we
end up with ~48 carbons and ~28 DoU left for aromatic and
aliphatic (0-45 ppm) structures. If we assume that all rings
are benzene types this requires
2
/
3
DoU pr. carbon. However,
if also heterocyclic aromatic rings are present like furans,
benzofurans etc. it is closer to DoU pr. carbon. 28 DoU
corresponds in the case of
2
/
3
DoU pr. carbon to 42 carbons
and in the case of DoU pr. carbon to 37 carbons of aro-
matic type leaving only 6 respectively 11 carbons for the
O
OH
O
OH
O O
OH
O
O
O
CH
3
CH
3
OH
O
OH
O O
N H
O
C H
3
O
OH
a)
OH
O
O
CH
3
O H
O
O
OH
O
O
OH
O
C H
3
OH O
O
OH
CH
3
O
OH O H
O
OH
C H
3
O
CH
3
O H
O
O
O
O
OH
O
O H
OH
O
O
C H
3
O
O H
OH
O
O
O
O H
O
C H
3
O
O
OH
O
OH O
O
OH
O
OH
O
b)
OH
SH
OH
O H
OH
O
OH
O H
O
O O
O
OH
O
O H
O
O
N H
2
OH
OH
O
OH
O
OH
OH
OH
OH
O
O H
OH
O
O
OH
O H O
c)
O
O O
OH
O H O
O H
OH
O H O H
O
O
O H
O H
O
O
OH
O H
O
O
OH
OH
O
O
OH
O
OH
CH
3
NH
O H
O H
O
O H
O
O H
O H
OH
O H
O
O
OH
OH
O
OH
O
O H
O
O H O
CH
3
O
OH
O
d)
O H
O
C H
3
CH
3
OH
O
O OH
O H
O
OH
O
OH
CH
3
N H
2
O NH
NH
O
S H
O NH
CH
3
CH
3
CH
3
OH
O H
O
OH
O
OH
C H
3
O
OH
C H
3
O H
O
OH
O
OH
C H
3
O H
P
O O H
O
OH
O
O H
O
OH
O H
O
OH
O
N
H
O H
O
O H
OH
O
OH O
O
O H
OH
C H
3
CH
3
OH O
OH O
O
O H
OH
C H
3
CH
3
O
OH
OH
O H
OH
O H
e)
aliphatic region. Integration of liquid state
13
C-NMR gave
18% aliphatic (0-45 ppm) carbon [10] corresponding to 11.7
aliphatic carbons in the proposed structure with totally 65
carbon atoms. Since only 6-11 carbons were left to aliphatic
structures in the above calculation, this even leaves room for
a number of non-aromatic ring structures. Furthermore it
shows that the aliphatic part in humic acids of field type is
most likely not underestimated using liquid state
13
C-NMR.
On that basis we have decided to use this NMR method for
our reconstructions or in other words as our tool to evaluate
the various models. Another good reason for this is the nar-
rower line widths of liquid state spectra, and hence more
insight into the presence of various structural elements (Fig.
4), as well as the possibility to distinguish amides/esters
from carboxylic acids [10].
Spectra of various HAs and FAs are presented in Figs. (5
and 6). The presented spectra are typical for a range of HAs
and FAs, and should largely cover those fractions and envi-
ronmental compartments that the HS-models in Figs. (2 and
3) are suggested to represent. Spectra of HA from widely
different soils are somewhat similar, reflecting that despite
the differences in parent material, HS-fractions from differ-
ent geographical and environmental compartments are most
often surprisingly similar. This is the case regarding struc-
tural elements as well as various physical behaviors. Also
between soil horizons, the differences can be quite minimal,
as seen for forest HA extracted from the organic horizon
(Fig. 5d) and from a subhorizon (Fig. 5e). PCA analyses
have previously demonstrated HAs extracted from lignite,
like Aldrich HA, to be in a class of their own when com-
pared to soil HA [9, 10]. This is due mainly to the lack of
some specific groups like amino acids and carbohydrates
(Fig. 5a), but despite this, Aldrich HA does share some simi-
larities with the field and forest HAs. The marine HA (Fig.
5f), although less aromatic than the other HAs, also shares
most of the structural elements of the soil HAs.
The FA-fractions (Fig. 6) are in general less aromatic
than the HA-fractions from the same environments. Beside
this, they do however carry most of the same structural ele-
ments and show somewhat similar shapes of the
13
C-NMR
spectra, also when comparing the terrestrial FAs (Fig. 6a &
b) with the aquatic FA (Fig. 6c). Significant differences are
lower amounts of amino acids in the FA-fraction (seen as a
less pronounced peak ~55 ppm) and a shift away from long
chain unbranched aliphatics (seen as a peak ~30-35 ppm)
towards peaks in the aliphatic area ~40 and/or 20-30 ppm.
This does not really seem to be the case for the Suwannee
River FA, though (Fig. 6c).
Simulated
13
C-NMR Spectra of HS-Models
Predicted
13
C-NMR spectra of all 11 HS-models in Figs.
(2 and 3) are presented in Figs. (7 and 8).
Humic Acids
The authors who suggested the molecularly large soil HA
polymer model in Fig. (2a) did not ascribe their model to HA
from any specific environmental compartment [21]. Since
the model lacks amino acids and carbohydrates, it is not im-
mediately a very good model for soil HAs. This is clearly
reflected in the simulated spectrum (Fig. 7a) and further-
more, there seem to be too many aliphatic subunits and too
few aromatic and carboxylic subunits. Also, the aromatic
area is too narrow around the typical chemical shifts of un-
substituted aromatic carbons, which indicates too few O-
substituted aromatics compared to typical HAs. Neverthe-
less, compared to any of the other model simulations, this
model does show the best fit in the unsubstituted aromatic
region (115-145 ppm), with a clear peak around 128 ppm,
which is typically observed in
13
C-NMR spectra of both HAs
and FAs. HAs from lignite do not contain significant
amounts of amino acids and carbohydrates, and in compari-
son with Aldrich HA (Fig. 5a), the simulated
13
C-NMR spec-
trum of the model proposed by Schulten & Schnitzer [21] is
rather good. Looking at other properties of the model, the fit
of this HA is less perfect, with regards to e.g. the CHNO
content, which is far from lignite HAs like Aldrich HA (Ta-
ble 1), and also different from what is typically observed for
soil HAs [1, 34], Table 1.
The model proposed by Stevenson [1], contains amino
acid and carbohydrate structural elements and therefore
could be a likely candidate for soil HA, but as quickly rec-
ognized from the simulated spectra there are some deficien-
cies in the aliphatic part of the spectrum. The rather recent
models proposed by Diallo et al. [13] and Albers et al. [10]
are both constructed to be models of soil HA. While the
models are clearly better representatives of soil HA than
many other published models, they also have some deficien-
cies. In the Albers et al. HA, despite having the right propor-
tions with regards to various structural elements and also the
elemental content (Table 1) [10], some aliphatic signals in
the region 30-35 ppm are missing. Furthermore, the aromatic

Fig. (4). Comparison of a) liquid and b) solid state
13
C-NMR spectra of a forest HA. Peaks assigned according to Albers et al., 2008 [10].
a)
Aliphatic
Amino acid
C
C
1
C
2-5
C
6
ArCH
ArCO
Amide/ester
C=O
COO
-
Carbohydr.
200 180 160 140 120 100 80 60 40 20 0 ppm
Aliphatic
Amino acid
C
C
1
C
2-5
C
6
ArCH
ArCO
Amide/ester
C=O
COO
-
Carbohydr.
200 180 160 140 120 100 80 60 40 20 0 ppm 200 180 160 140 120 100 80 60 40 20 0 ppm
b)
200 180 160 140 120 100 80 60 40 20 0 ppm 200 180 160 140 120 100 80 60 40 20 0 ppm


Fig. (5). Liquid state
13
C-NMR spectra of humic acids (HAs). a) Aldrich HA, commercial HA extracted from lignite. b) Field HA, extracted
from clayey soil. c) Field HA, extracted from the B
h
-horizon of an acidic sandy podzol. d) Forest HA, extracted from the organic H-horizon.
e) Forest HA, extracted from the B
h
horizon of a forest podzol. f) Marine HA [32].

Fig. (6). Liquid state
13
C-NMR spectra of fulvic acids (FAs). a)
Field FA, extracted with acid from the B
h
-horizon of an acidic
sandy podzol. b) Grassland FA, extracted from a clayey soil. c)
Aquatic FA, the IHSS standard FA from Suwannee River [33].
region is not shaped, with a clear peak around 128 ppm, as
seen for recorded spectrum of soil HA. The same is the case
for the model proposed by Diallo et al. [13] and furthermore
this model clearly lacks aliphatic structures which will give
rise to chemical shifts around 30 ppm. Likely candidates for
this would be simple unsubstituted aliphatic chains, which
might be incorporated in the proposed model. Furthermore,
as previously pointed out [10], the N- and S-content should
be changed in order to make this model in better agreement
with typical soil HAs.
Sein et al. [22] proposed a model of terrestrial HA (Fig.
2e), but it was not clear if the HA used as a model HA, was
derived from soil or lignite. The proposed model could pos-
sibly be one structural element in HA, since, apart from a too
intense carbonyl-signal, its simulated spectrum contains no
wrong signals (Fig. 7e). It does however miss amino acids,
which is reflected in the lack of signal ~55 ppm and further-
more it contains no unsubstituted aliphatics, which is also
reflected in the simulated spectrum as the lack of signal be-
low ~32 ppm. It can therefore not stand alone as a represen-
tative structure of HA.
We have included a model of a marine HA, since, com-
pared to soil HAs, these HAs have very different parent ma-
terial, which would also be expected to be reflected in their
final structure and hence
13
C-NMR spectra (Fig. 5f). One of
the characteristics of marine HAs is their low aromatic con-
tents, and this feature is well captured by the model of Har-
vey et al. [23]. Furthermore, the model contains rather long
aliphatic chains, which give rise to the intense signal at ~30
ppm, which is also seen in the recorded
13
C-NMR spectrum.
a)
Aldrich HA
220 200 180 160 140 120 100 80 60 40 20 ppm
Aldrich HA
220 200 180 160 140 120 100 80 60 40 20 ppm
b)
Clay field HA
220 200 180 160 140 120 100 80 60 40 20 ppm
Clay field HA
220 200 180 160 140 120 100 80 60 40 20 ppm

c)
B
h
field HA
220 200 180 160 140 120 100 80 60 40 20 ppm
B
h
field HA
220 200 180 160 140 120 100 80 60 40 20 ppm
d) 220 200 180 160 140 120 100 80 60 40 20 ppm
H-hor. forest HA
220 200 180 160 140 120 100 80 60 40 20 ppm
H-hor. forest HA
105
e) 220 200 180 160 140 120 100 80 60 40 20 ppm
B
h
-hor. forest HA
220 200 180 160 140 120 100 80 60 40 20 ppm 220 200 180 160 140 120 100 80 60 40 20 ppm
B
h
-hor. forest HA
f)
Marine HA
220 200 180 160 140 120 100 80 60 40 20 ppm
Marine HA
220 200 180 160 140 120 100 80 60 40 20 ppm

a)
B
h
field FA
220 200 180 160 140 120 100 80 60 40 20 ppm
B
h
field FA
220 200 180 160 140 120 100 80 60 40 20 ppm

b)
Grassland FA
220 200 180 160 140 120 100 80 60 40 20 ppm
Grassland FA
220 200 180 160 140 120 100 80 60 40 20 ppm

c)
220 200 180 160 140 120 100 80 60 40 20 ppm
Suwanee River FA
220 200 180 160 140 120 100 80 60 40 20 ppm
Suwanee River FA

The two distinct peaks at ~55 and 63 ppm, attributed to
amino acid C and C
6
of carbohydrates, respectively, is
however not seen in the simulated spectra, and looking at the
model structure (Fig. 2f), no carbohydrate or amino acid
structural elements are seen. Since also a peak at ~105 ppm
(anomeric carbohydrate signal) is seen in the recorded spec-
trum of a marine HA, most of the signal around 75-80 ppm
most likely derives from carbohydrate, and the model should
probably contain less aliphatic alcohols and instead include
carbohydrate structures. Furthermore, there are four ketonic
carbons in the model, which gives rise to a rather intense
signal around 205-210 ppm, while the ketonic signal in the
recorded spectrum is low.
Fulvic Acids and Whole Soil HS
The simulated spectra of the four FA-models and the sin-
gle HS-model are shown in Fig. (8). The soil FA model pro-
posed by Shin & Moon [24], is a further development of
models previously proposed for Suwanee River FA and the
simulated NMR-spectra (Fig. 8a) is not too different from
the Suwanee River FA model, proposed by Leenheer &
Rostad [25] (Fig. 8b). No amino acids are included in neither
of these models, which is immediately seen as the lack of
signal ~55 ppm where you would usually see a signal from
amino acid Cs. Aquatic FAs often do not contain signifi-
cant amounts of amino acids but, although in lower amounts
than the corresponding HA fractions, terrestrial FAs most
often do and amino acids should probably be included in this
model. The lower amounts of long-chain aliphatics is re-
flected in a less pronounced peak ~30-35 ppm, in Fig. (8a &
b). The shape of the aliphatic area in Fig. (8b) fits well with
the recorded spectrum of some soil FA-fractions (Fig. 6b),
but it fits the aquatic FA (Fig. 6c), which it was constructed
for, less well.
Furthermore the intense peak at ~170 ppm (derived from
esters in the model) is not really seen in any of the recorded
spectra for FAs. The Suwanee River FA seems to be an illus-
trative example, where an integration of the areas assigned to
certain overall structural elements (e.g. aromatics or aliphat-
ics) would lead to the conclusion that the model fits very
good, while looking at the simulated spectra, it is obvious
that the specific nature of the structural elements, cannot be
more than partly correct. This is also the case for the model
of terrestrial FA proposed by Alvarez-Puebla et al. [26]), in
which the aliphatic fraction of C-atoms is so branched and
substituted with O-atoms, that, besides being too small over-
all, it shows most of the aliphatic signals >60 ppm and no
aliphatic signals <40 ppm, which is obviously a deficiency.
This shape in the aliphatic area is more pronounced but still
somewhat similar to the HA proposed by Sein et al. [22]
(Fig. 2e & 7e), which was actually the basis for developing
this model of FA [26].
The FA-model proposed by Albers et al. [10] (Fig. 8d)
reveals the best shape compared to the recorded spectra, es-
pecially to the grassland FA in Fig. (6b). The shape of the
aromatic peak is not perfect though, with signals missing
especially ~130 ppm. This is probably caused by the fact that
no unsubstituted aromatic C-atoms exist in the model (Fig.
3d). Furthermore, some fill-in between aromatic peaks is
missing in general, which supports the concept that a puri-
fied HS-fraction is a mixture of several more or less similar
molecules.

Fig. (7). Simulated
13
C-NMR spectra of models of humic acids
(HAs). The units on the x-axes are all in ppm. a) Not further de-
fined HA (Schulten & Schnitzer, 1993) [21]. b) Not further defined
HA (Stevenson, 1994) [1]. c) Agricultural soil HA (Albers et al.,
2008) [10]. d) Agricultural soil HA taken from Diallo et al. (2003)
[13]. The spectrum was based on five proposed substructures of
which one is shown in Fig. (2d). e) Terrestrial HA from commer-
cial Acros Organics Humic Substance (Sein et al., 1999) [22]. f)
Marine HA (Harvey et al., 1983) [23].
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
a)
b)
c)
d)
e)
f)

Fig. (8). Simulated
13
C-NMR spectra of fulvic acids (FAs) and
whole soil HS. The units on the x-axes are all in ppm. a) Soil FA
(Shin & Moon, 1996) [24]. b) Four structural units representing
Suwannee River FA (Leenheer & Rostad, 2004) [25]. c) Terrestrial
FA from commercial Acros Organics Humic substance (Alvarez-
Puebla et al., 2006) [26]. d) Agricultural soil FA (Albers et al.,
2008) [10]. e) Assembly of the partial structures included in a con-
ceptual model for whole soil SOM (Kleber et al., 2007) [27].

The model proposed by Kleber et al. [27] is a model of
SOM in general, that is both FA, HA, humin as well as non-
HS SOM, the latter typically making up 25-60% of SOM [1,
2]. The simulated spectrum of this model can therefore not
be expected to make up more than ~50% of a mixed spec-
trum of HA and FA. We have nevertheless included this
model, since it is the first model constructed according to the
supramolecular structural view of HS/SOM. Furthermore,
since it is a model of SOM, which includes HA and FA, it
should be possible to choose parts of the model, which
would then fit the recorded spectra of HA and/or FA. Since
the model contains no O-substituted aromatic C-atoms, this
is however not possible, and the simulated spectrum is
quickly recognized as very different from the recorded spec-
tra of HA and FA. Since we use liquid state
13
C-NMR as our
tool to evaluate models of HA and FA, it is not wise to make
too many conclusions of this SOM-model. We can neverthe-
less say, that it will be extremely difficult to choose struc-
tural elements of the SOM-model that will fit either HA- or
FA-fractions, which are, after all, important fractions of
SOM, quantitatively as well as regarding several properties
of SOM. This conclusion would be supported by an elemen-
tal analysis, which for this model would give 64% C, 8% H,
1.7% N and 24% O (Table 1) and a titration of acid groups,
which would also reveal the lack of phenolic structures,
which are always part of the HA- and FA-fraction of SOM.
Kleber et al. [27] have some very interesting discussions on
organo-mineral interactions on soil, but the model, which
they propose, seems to contain very little of the compounds,
which would normally be extracted into the HA- and FA-
fractions.
GENERAL REMARKS
The advantage of using a
13
C-NMR simulation is that all
carbons within a structure are taken into account, and can be
immediately compared to recorded NMR-spectra. Although
several overlaps will occur in a 1D-NMR spectrum of humic
substances and it therefore not always is possible to be con-
clusive on the exact structural elements, the inclusion of all
structural elements makes possible a better balance between
the various parts of the spectrum. This balance should of
course be checked in relation to an elemental analysis. A
titration of acid groups to divide these into carboxylic acids
and phenols likewise makes a good check when compared to
the NMR results. Finally, both nitrogen and in the case of
lignite HS also sulphur content play a role. The former can in
some cases, when being part of amino acids, be correlated to
the NMR findings [10].
The aromatic region in general plays an important role as
many humic substances contain large proportions of aro-
matic carbons. The very broad region assigned to aromatic
structures, clearly shows that a vast combination of struc-
tures are required not the least to explain the low frequency
wing (100-115 ppm) generally found in
13
C-NMRspectra of
HS-fractions. In HS-fractions containing significant amounts
of carbohydrates, this region can to some extent, but not
fully, be ascribed to anomeric carbons of carbohydrates
(~105 ppm). The oxygen substituted aromatic carbons can to
a good degree be determined as some of these signals will
appear around 160 ppm. To help understand, which aromatic
structures are present in a given HS-fraction, a check be-
tween oxygen content, number of titrated phenolic and car-
boxylic acid groups and NMR parameters, is necessary since
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
0 20 40 60 80 100 120 140 160 180 200 220
a)
b)
c)
d)
e)
part of the oxygen substituted aromatic carbons are buried in
the main signal due to structural elements as exemplified in
Fig. (1f). Parameters such as bulk densities and solubility
could also be taken into account [13]. Mass spectroscopic
studies are clearly very useful. Diallo et al. [13] showed that
most of the fragments of soil HA do not exceed 1200 Dalton.
This fits well with most of the proposed structures evaluated
in this paper, which are all below 1650 Dalton except the HA
proposed by Schulten & Schnitzer (1993), which is signifi-
cantly larger (~5800 Dalton). The simulated spectra have
shown that even with very broad line widths, the resulting
spectra of such small molecules do always miss some fill-in,
especially in the aromatic region. This supports the idea that
even a purified HS fraction is a mixture of several molecules
and that one final structure of a HA or FA cannot be identi-
fied.
As indicated here, there are numerous considerations,
which have to be taken into account before it is possible to
construct a reasonable model structure, which makes a good
representation of either whole soil SOM or of selected frac-
tions like HA and FA. In the present paper, apart from hav-
ing evaluated some existing proposed models, we have
shown that using simulated
13
C-NMR spectra, in the process
of constructing such model structures, will help avoiding
wrong structural elements and hopefully likewise be a help
in choosing the right ones.
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Received: January 15, 2010 Revised: February 02, 2010 Accepted: February 02, 2010

Albers and Hansen et al.; Licensee Bentham Open.


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The Open Magnetic Resonance Journal

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