Structure and Dynamics of Hydrogels and Organogels An NMR

Progress in Polymer Science 36 (2011) 11841253
Contents lists available at ScienceDirect

Progress in Polymer Science
j our nal home page: www. el sevi er . com/ l ocat e/ ppol ysci
Structure and dynamics of hydrogels and organogels: An NMR
spectroscopy approach
Yury E. Shapiro
Mina and Everard Goodman Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
a r t i c l e i n f o
Article history:
Received 23 September 2010
Received in revised form7 April 2011
Accepted 7 April 2011
Available online 19 April 2011
Keywords:
Hydrogels
Organogels
NMR spectroscopy
Network structure
Morphology and mobility
a b s t r a c t
Hydrogels and organogels are semi-solid systems, in which a liquid phase is immobilized
by a three-dimensional network composed of self-assembled, intertwined polymer/gelator
bers. Investigations pertaining to these systems have only picked up speed in the last
few decades. Consequently, many burning questions regarding these systems, such as
the specic molecular requirements guaranteeing gelation, still await denite answers.
Nonetheless, the application of different hydrogels and organogels to various areas of
interest, i.e., as drug delivery devices, has been quick to follow their discoveries.
The use of NMR spectroscopy for the characterization of polymer hydrogels and
organogels has recently seen enormous growth. The NMR measurements involving magic
angle spinning (MAS) in the solid-state NMR, spin relaxation times, nuclear Overhauser
enhancements (NOE), or multiple-quantum (MQ) spectroscopy, the pulse eld gradient
(PFG) technique and magnetic resonance imaging (MRI) allow obtaining the detailed infor-
mation on morphology, molecular organization, specic interactions and internal mobility
of constituents.
This review aims at providing a global view and capabilities all of these NMR methods in
comprehensive studies of hydrogels and organogels, with special emphasis onthe interplay
between the morphology and molecular mobility of constituents and the intermolecular
interactions.
2011 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
1.1. Hydrogels and organogels: denition and history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
1.1.1. Hydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186
1.1.2. Organogels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186
1.2. Properties of hydrogels and organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1186
1.3. NMR spectroscopy of gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
1.3.1. General experimental approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
1.3.2. Spinlattice and spinspin relaxation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
1.3.3. Chemical shift and its anisotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
1.3.4. Self-diffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
1.3.5. Concepts of the nuclear Overhauser enhancement (NOE). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
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E-mail address: shapiro@nmrsgi4.ls.biu.ac.il
0079-6700/$ see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.progpolymsci.2011.04.002
Y.E. Shapiro / Progress in Polymer Science 36 (2011) 11841253 1185
1.3.6. Main sources of NMR signal broadening in gels and solids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191
1.3.7. Experimental tools for NMR spectroscopy in hydro-/organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1191
1.3.8. (
1
H)
13
C cross-polarization under MAS conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
1.3.9. The solid-state
2
H NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
1.3.10. Relaxation of I = 1/2 nuclei in the rotating frame . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
1.3.11. Magnetic resonance imaging (MRI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192
2. NMR spectroscopy on the mechanisms of gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1193
2.1. Gelation and gelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1193
2.2. Sol and gel phases as detected by NMR spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1193
2.3. Self-assembly of brils and morphology of gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197
2.3.1. Self-assembly of hydrogel network from polymers and oligomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1197
2.3.2. Self-assembly of LMWG networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1198
2.3.3. Networks based on the inclusion complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1199
2.3.4. Stereochemical control of the self-assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1201
2.3.5. Thermodynamics of gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1202
2.4. Hydration and hydrogen bonding in gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1203
2.5. stacking and van der Waals interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1208
2.6. Chemical crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
2.6.1. Model studies of the initial stage of crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
2.6.2. Estimation of the crosslinking degree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211
2.6.3. Mechanisms of crosslinking by gelation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215
2.6.4. Swelling and deswelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1218
3. NMR spectroscopy on the molecular dynamics of hydrogels and organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1219
3.1. NMR relaxometry and gel dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1219
3.1.1. General consideration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1220
3.1.2.
1
H T
2
relaxation affected by an exchange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
3.1.3. Anisotropic molecular motion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
3.1.4.
1
H T
1
relaxation affected by an exchange. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221
3.1.5.
1
H T
1
for polymers with no exchangeable protons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
3.2. NMR relaxometry on discrimination and self-assembly of species with different mobility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1222
3.3. NMR relaxometry on network structure and crosslinking reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1226
3.4. Detection of slow mobility in gels with one-dimensional exchange NMR experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1228
3.5. PFG-NMR on self-diffusion of constituents of hydro-/organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1229
3.5.1. Pulsed eld gradient spin-echo experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1230
3.5.2. Diffusion models in gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1231
3.5.3. Self-diffusion of a solvent in hydro-/organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1231
3.5.4. Self-diffusion of low- and high-molecular weight compounds in hydro-/organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . 1234
3.5.5. Self-diffusion of the hydro-/organogel network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237
4. NMR spectroscopy on the morphology of hydrogels and organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237
4.1. NOE measurements for study of hydro-/organogel morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1237
4.2. NMR diffusometry on morphology of hydro-/organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1239
4.3. NMR cryoporometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1242
4.4.
129
Xe NMR in the porosity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1242
4.5. MRI of hydrogels and organogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1243
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1245
1. Introduction
A recent issue in supramolecular chemistry of smart
materials is the focus on the organization of monomer and
polymer species into desired superstructures. Hydrogels
and organogels belong to these materials and are charac-
terized by more than one length scale through crosslinks
formed by covalent bonds or physical (hydrogen bonding,
solvophobic, charge transfer and van der Waals) interac-
tions.
NMR spectroscopy in combination with FTIR, X-ray,
electron microscopy and others, is rewarding in study of
morphology, molecular structure and component dynam-
ics of gel networks. For example, the chemical shifts
and intensities of peaks in the NMR spectra allow
structural quantities such as polymer composition, micro-
tacticity, sequence distribution, branching, crosslinking
and molecular weight to be measured [14], while the
more sophisticated experiments, i.e., magic angle spinning
(MAS), measurements of spin relaxation times, nuclear
Overhauser enhancements (NOE), or multiple-quantum
(MQ) spectroscopy, as well as pulse eld gradient (PFG)
technique and magnetic resonance imaging (MRI) can pro-
vide detailed information about molecular organization,
specic interactions and internal mobility of constituents
[5,6].
1.1. Hydrogels and organogels: denition and history
We begin with denitions of a fewterms used through-
out this review[7].
1186 Y.E. Shapiro / Progress in Polymer Science 36 (2011) 11841253
Sol: The term refers to a colloidal system of liquid char-
acter in which the dispersed entities are either solid
nanoparticles or molecules (gelators). Some sols can
be transformed into gels by increasing concentration
of the dispersed substance above the so-called crit-
ical gelator concentration (CGC), or decreasing the
temperature down to the sol-to-gel transition tem-
perature, T
SG
.
Gel: A gel is a colloidal systemof solid character in which
the dispersed substance (polymer, low-molecular
gelator or their combination) forms a continuous,
ramifying, coherent framework that is interpene-
trated by a system(usually liquid) consisting of units
smaller than colloidal entities. Gels combine rigidity
with elasticity [8] being unique materials, which are
well known in daily life and have a broad range of
applications in food, medicine, biomaterial, cosmetic,
separation, etc., technologies [9]. The majority of the
material consists of uid solvent and only a minor-
ity of solid matrix is present [10]. The classic gel has
typically less than 10% of a crosslinked polymer, or
less than 1% of the low-molecular weight gelator. As
a result, a large solidliquid interfacial area is present
within the gel, where solutes can be entrapped in the
pores formed by the solid component.
Therearethreetypes of gel systems distinguishedbythe
kind of the network-forming gelator: made of a polymer
gelator, of the self-assembled low-molecular weight gela-
tor (LMWG) and prepared in the course of self-assembly of
polymer and surfactant, or some other LMWG. Gelators are
a class of molecules that can undergo self-organization in
a particular solvent to yield a ne brous structure.
Gels have been eloquently described as being the
result of crystallization gone awry [11]. Indeed, macro-
scopic phase separation into crystalline and liquid
layers is avoided in these systems owing to the bal-
ance between gelator aggregating forces and solubilizing
solventaggregate interactions. The overall thermody-
namic and kinetic gel stability results from the interplay
of the opposing forces related to the gelator partial solu-
bility in the continuous phase. Normally, two types of gels
are distinguished according to the nature of liquid phase:
organogels, containingorganic solvent, andhydrogels, con-
taining water.
1.1.1. Hydrogels
Hydrogels are materials with three-dimensional, water-
swollen structures composed of mainly hydrophilic
polymers, oligomers or roads, self-assembledfromLMWGs
[12]. They are rendered insoluble due to the presence of
chemical or physical crosslinks. The physical crosslinks
can be entanglements, crystallites, or weak associations
formed by van der Waals interactions, -stackings or
hydrogen bonds. The crosslinks provide the network struc-
ture and physical integrity.
Since the development of poly(2-hydroxethyl
methacrylate) hydrogel in 1960s, hydrogels have been
considered for use in a wide range of biomedical and
pharmaceutical applications due to their high water con-
tent and, hence, excellent biocompatibility [9,10,1214].
Some applications of hydrogels include contact lenses,
biosensors, sutures, dental materials, and controlled drug
delivery devices. The network permeability and swelling
behavior of hydrogels are the most important character-
istics in evaluating their ability to function in a particular
controlled drug release and protection applications. They
are strongly dependent on the chemical nature of the
polymer/LMWG composing the gel as well as the structure
and morphology of the network, which is subject to NMR
investigations.
The so-called stimuli-responsive hydrogels show dras-
tic changes in their properties in response to the external
pH, temperature, ionic strength changes or nature of
swelling agent, and irradiation, and belong to the so-
called smart materials applicable for actuators, sensors,
switching devices, etc. [1315]. Whenthe driving forces for
the gelation involve only physical crosslinks, the resulting
hydrogels are found to be thermoreversible. For instance,
polymers based on polyacrylic and polymethacrylic back-
bones show this behavior. On the other hand, the
gels formed by chemical crosslinking (e.g., polyesters,
polyamides, etc.) generally are not thermoreversible [16].
1.1.2. Organogels
Organogels can be distinguished from hydrogels by
their predominantly organic solvent. The gelation process
involves the self-assembly of LMWGs to give polymer-like
bers, which immobilize the organic solvent by form-
ing a three-dimensional network of either crosslinked or
entangled chains for chemical and physical gels, respec-
tively. The bers are stabilizedby weak interactions suchas
hydrogen bonding, van der Waals forces, stacking and
hostguest assembly [8,11,17]. Many factors such as steric
effects, rigidity, and polarity can counter the self-assembly.
Control over the gelation process as well as the conception
of new gelling molecules remain important challenges to
face in the quest of new organogelators. Up to date, it is
known only that a gelator has to bear any conventional
gelatingmotif suchas amides, longalkyl sidechains, steroid
groups [17], and so on. However, a few of LMWGs were
discovered, which do not have these motifs [18].
Though the many chemical compositions and physical
properties of gels are known yet, several principal ques-
tions remain to be answered [11]. Among these are: (i)
what are the structural requirements for a molecule to gel
an organic liquid? (ii) What is the relationship between
the packing arrangements of gelator molecules in their
bulk crystalline states and in their various aggregates in
the gels? (iii) How does the molecular packing of gelator
molecules affect the mechanical, thermodynamic, optical,
and other properties of the gels? These and other basic
questions relate to a practical goals to discern a priori
whether and under what conditions anorganogel will form
andcanbeansweredwithaidof various structural methods
of analysis including profoundly NMR spectroscopy [19].
1.2. Properties of hydrogels and organogels
The rod-like elementary assemblies (crystalline bers,
tapes, strands, etc.) are rather efcient by immobilization
of a large amount of liquid by a small quantity of gela-
tor [11]. These elongated objects link with each other at
junction zones to form three-dimensional sponge-like
network that entraps a solvent by capillary forces and sur-
face tension [20]. The structure of linear aggregates in the
gel is determined by the direction and strength of the
binding forces associated with the aggregation process.
In aqueous solutions of amphiphiles, aggregate structures
result from a balance between the attractive (interfacial)
and repulsive forces (between head groups) [21]. In non-
aqueous liquids, the attractive forces are mainly dipolar
interactions and, possibly, specic intermolecular hydro-
gen or metal-coordination bonds. They must balance the
free energy increase that accompanies reductions in trans-
lational and rotational freedomof motion.
Organogels canexhibit either permanent rigidnetworks
or transient semiexible meshes. The notion of transient
meshsize or statistical porosity of a three-dimensional net-
work is similar to the description of polymeric solutions
(with blobs of size ) when individual chains interact
with each other [22]. Instead of the solid component sepa-
rating in crystals, the ratio of whose dimensions is almost
invariant with overall size, bers are formed, in which the
cross-sectional dimensions arenearlyconstant irrespective
of the overall aggregate length. Some structural and kinetic
aspects of the aggregation/gelation process are also consis-
tent withits being viewedas a phase separationinvolving a
nucleation reaction, thus emphasizing the role of the inter-
facial free energy of the gelators (in contrast to spinodal
decomposition phenomena or unwinding processes).
Rheological properties appear to be the best diagnos-
tics to follow the changes in gelator aggregation during
the sol-to-gel (self-assembly) and gel-to-sol (melting)
phase transitions [23]. They can be described by statisti-
cal physics, and, in particular, percolation models [24,25]
cansimulatethemechanical behavior near thecritical gela-
tion threshold. The sol-to-gel phase transition is generally
characterized by a divergence of the connectivity corre-
lation length [26] (as is the case for second-order phase
transitions).
Two different types of three-dimensional networks
and viscoelastic materials can be obtained in organogel
systems. In the rst, materials exhibiting a solid-like, vis-
coelastic mechanical behavior and which are made of
permanent networks obtained through a sharp sol-to-
gel phase transition at a specic temperature T
SG
are
strong gel-like systems. In the second, materials exhibit-
ing a liquid-like viscoelastic behavior which are made of
transient networks (obtained through a much smoother
threshold) are termed weak gel-like systems. Both
types of networks are thermally reversible. On heating,
the supramolecular architecture is melted and individual
molecules (or separated strands of aggregated molecules)
are re-dispersed in the bulk solution.
Three fundamental predictions of rubber elasticity the-
ory [27,28] are applicable for gels and relate the average
molecular weight betweencrosslinks <M
c
> to the volumet-
ric swelling ratio Q, the elastic modulus G
, and crosslink
density
e
, which followscaling relationships of the form:
Q
v
-M
c
:
3]5
, C
-M
c
:
1
and
e

P
-M
c
:
, (1)
<M
c
>,
Crosslinks
Fibers Fibers
Fig. 1. Schematics of the crosslinked bers in a gel. <M
c
> is the molecular
weight of the polymer chains between crosslinks, and is the network
mesh size.
where
P
is the density of the polymer, and <M
c
> can
be approximated from the FloryRehner equation [28].
Other measures of gel properties include mesh size and
crosslink density
c
. These obey the following scaling rela-
tionships:
c
-M
c
:
1
and -M
c
:
1]2
. (2)
The structure of an idealized gel as a network of the
crosslinked bers is shown in Fig. 1. The most impor-
tant parameters that dene the structure and properties
of swollen gels are the polymer volume fraction, <M
c
>,
and [12]. These parameters, which are not independent,
can be determined theoretically [28] or through a variety
of experimental techniques, including NMR spectroscopy
[29]. Fig. 2 demonstrates the elements of gel morphol-
ogy based on the typical TEM image of a freeze-dried
-manno/p-xylene organogel [30]. The TEMimage is very
similar to those for other organogels and hydrogels, so we
can consider this image as a common feature.
Several models havebeendevelopedtoexplainthetran-
sition frommolecular to primary and secondary structure
of bers [31]. For example, Bodenandco-workers [32] have
modeled the hierarchical self-assembly of rod-like chiral
molecules, such as peptides in a -strand conformation,
into ribbons and bers (Fig. 3). They begin with a chi-
ral rod-like monomer functionalized with complementary
donor and acceptor groups on opposite sides and chem-
ically different surfaces (in Fig. 3a, black=hydrophobic,
white =hydrophilic). In solution, the rods rst assemble
into one-dimensional tapes via recognition of the donor
and acceptor groups (Fig. 3c). The chirality of the monomer
is translated into the twist of the tape, and the two sides
of the tape are chemically different, resulting in different
afnities for the solvent. This in turn leads to a helical cur-
vature to the tape (Fig. 3c
). The tapes can further assemble,

due to greater attraction between the hydrophobic (black)
faces, into ribbons (Fig. 3d). Now both sides of the ribbon
are chemically equivalent, resulting in a saddle-like cur-
vature (Fig. 3d
). Subsequent aggregation (with increasing

concentration) leads to the formation of brils and then
bers (Fig. 3e and f). The chirality-induced twist leads to
Fig. 2. (a) Chemical structure and molecular size of the
mannose-appended organogelator methyl-4,6-O-benzylidene--d-
mannopyranoside (-manno). (b) Typical TEM image of a freeze-dried
sample prepared from the -manno/p-xylene gel. The TEM image
suggests that the 100nm diameter ber is the constitutional unit and
those bers are bundled together to make thicker bers. The bers cross
and stick together to make the junction point. The blank region had been
occupied by the solvent before the sample was freeze-dried. This TEM
image is very similar to those for other organogels, so we can consider
that this image is a common feature for organogels [30]. Copyright
permission fromAmerican Chemical Society, 2003.
brils and bers with well-dened widths dependent on
balancing the favorable attractive interactions and unfa-
vored distortion energies fromthe twisting.
The role of the organic solvent in the gel formation
process remains ambiguous [11]. However, it was shown
that the enthalpies of gel formation are largely indepen-
dent of the solvent, but strongly dependent on the gelator
structure. It has, therefore, been suggested that the sol-
vent is entrapped between microcrystals of the gelator
by capillary forces [33]. Whitten and co-workers [34,35]
have addressed this matter with a detailed research on
the microstructure of the aggregates in the gel, formed by
p--cholesteryl-p
-octoxystilbeneoate in higher alcohols,

e.g., n-butanol and n-octanol by means of
1
H NMR, AFM
and uorescence microscopy. As a dynamic temperature-
dependent model, they proposed the existence of rigid gel
strands that are composed of large bril bundles, which are
probably both microcrystalline and devoid of solvent. With
increasing temperature, a solvent-induced ber swelling
of the internal bril bundles into mobile gel strands or
breakingupintosmaller moreexiblebril bundles occurs.
Finally, the tertiary structure of a gel involves the inter-
action of individual aggregates and ultimately determines
whether a gel is formed or, instead, bers precipitate from
solution rather than trap it. Physically, long, thin, exible
bers are better able than shorter bers to trap solvent
leading to gelation [31]. The transition from secondary to
tertiary structure is determined by the type of interactions
that canoccur among the bers. Gels canbe formedby both
physically branched bers (interconnected networks) and
entangled bers. The type of crosslinking determines the
rheological properties of the gel.
1.3. NMR spectroscopy of gels
The key to a structural analysis of hydro-/organogels
by NMR is the fact that NMR data render information
on the chemical nature as well as on the molecular or
collective mobility of an observed component [6,36,37].
Any observed chemical constituent of gel may therefore
be assigned to the denite phase depending on its indi-
vidual motional behavior. Whereas a liquid or dissolved
in the uid phase component will preserve its full dif-
fusive mobility depending on the overall viscosity, any
constituent of the polymer network will be substantially
immobilized within the gel matrix. In most cases attention
is paid to the analysis of the variation of chemical shifts,
spin relaxation times, or intensity of the NMR signals along
with concentration, solvent composition, or temperature.
Those data can be used to obtain information on the nature
of the intermolecular interactions, the critical concentra-
tion values, the change in the motion of the molecules,
or thermodynamic parameters associated with the gel
formation.
1.3.1. General experimental approaches
The principles of NMR spectroscopy are described else-
where [38,39] therefore only a fewgeneral statements are
collected here for the readers convenience. Magnetic res-
onance is based on the interaction between an external
magnetic eld and a nucleus that possesses spin I / = 0,
and, respectively, spin angular momentum. This angu-
lar momentum in the external static magnetic eld B
0
experiences the so-called Larmor precession about the
eld direction with the frequency of
0
=B
0
(where
is the magnetogyric ratio), allowing magnetization of the
nucleus. It is convenient to consider NMR experiments in
a rotating coordinate frame rather than in a xed labora-
tory coordinate frame. The rotating coordinate frame is one
in which the z axis is the B
0
eld direction, but the x- and
y-axes rotate about the z-axis with the frequency
0
. The
angular component due to the precession in the labora-
tory frame becomes xed in the rotating frame. After a 90
pulseof thesecondmagnetic eldB

1
, whichoscillates at the
appropriate (resonance) radio frequency (RF), the magne-
tization
i
of any magnetic nuclei i (
1
H,
2
H,
13
C,
15
N,
31
P,
etc.) in a sample, placed into magnetic eld B
0
, which was
aligned initially along z-axis inthe rotating frame, becomes
xedinthexyplane. This transversemagnetizationinduces
a signal decay shown in Fig. 4. Subsequent Fourier trans-
formation (FT) of the free induction decay (FID) yields the
spectrumS().
Fig. 3. Model of the hierarchical self-assembly of chiral rod-like monomers in hydro-/organogels. (a) The monomer with complementary functionality
and chemically different faces. The local arrangements (cf) and the corresponding global equilibrium conformations (c
) of the structures are formed

in solutions of the monomer with increasing concentration. See the text for details [32]. Copyright permission fromPNAS, 2001.
Fig. 4. Principles of NMR spectroscopy in hydro-/organogels (see comments in the text).
1.3.2. Spinlattice and spinspin relaxation
The equilibriummagnetization of a sample, M
0
=l
i
, is
aligned along the z-axis. If the spin systemis excited with
the RF pulse, this alignment is violated, andz-component of
magnetizationM
z
decreases. Inorder for the spinsystemto
return to equilibrium, there must be interaction between
spins and surroundings, or lattice, leading to a dissipation
of the excess energy. The rate at which the systemreturns
to equilibriumcan be described [38] by Blochs Eq. (3):
dM
z
dt
=
(M
z
M
0
)
1
1
, (3)
where the time constant T
1
is called the spinlattice relax-
ation time. It was shown that the spinlattice relaxation
rateR
1
depends onthefrequencydistributionof themotion
of a randomly tumbling molecule which is expressed in
terms of spectral density function J(
0
) as
R
1
=
1
1
1
8
2
xy
j(
0
) and j(
0
) =
z
c
(1 +
2
0
z
2
c
)
, (4)
where z
c
is called the correlation time and is a characteris-
tic time of isotropic molecular motion, and
0
is the Larmor
resonant frequency of the spin system.
Once disturbed from equilibrium, magnetization may
have components in the xy plane, and these will also relax
totheir equilibriumvalue of zero. The returntoequilibrium
is governed [38] by Blochs Eq. (5):
dM
xy
dt
=
M
xy
1
2
, (5)
where the time constant T
2
is called the spinspin relax-
ation time and is also a characteristic time of molecular
motion. The spinspin relaxation rate R
2
is
R
2
=
1
1
2
8
2
z
j(0) and j(0) = z
c
. (6)
The rate of the xy relaxation R
2
may be larger than, but
can never be smaller than, the rate of the z relaxation R
1
[40]. If FIDdecays rapidly(largeR
2
), thelineinthespectrum
is broad. The widthat half of the peak height is ^
1/2
=R
2
/
and, hence, proportional to z
c
if chemical shift anisotropy
and dipolar coupling are ignored.
1.3.3. Chemical shift and its anisotropy
In molecules, the resonance frequencies of various
chemical groups differ because of the different magnetic
shielding of nuclei caused by the surrounding electrons.
This phenomenon is known as the chemical shift, , given
in ppmas compared with a reference, which arises because
motions of electrons inducedby the static magnetic eldB
0
generate secondarymagnetic elds. The effect of secondary
elds, called nuclear shielding, can enhance or oppose the
main eld. In general, the electronic charge distribution
in a molecule is anisotropic, and effects of shielding on a
particular nucleus are described by the nuclear shielding
tensor withprincipal components o
xx
, o
yy
ando
zz
. Thelocal
magnetic eld at the nucleus is given [39] by Eq. (7):
8
loc
= (1 o
kk
)8
0
(7)
In isotropic liquid solution, collisions lead to rapid
reorientation of a molecule, and consequently, to averag-
ing the shielding tensor o =(o
xx
+o
yy
+o
zz
)/3. However, in
anisotropic liquids like liquid crystals and gels, the shield-
ing tensor does not experience averaging and we deal with
the chemical shift anisotropy (CSA) dened conventionally
as:
zo = o
zz
o
xx
+o
yy
2
. (8)
1.3.4. Self-diffusion
Macromolecules dispersed in a uid medium undergo
two distinct types of the overall Brownian diffusive
motions: translational self-diffusion and rotational tum-
bling [41]. In the case of solid spherical particles, the
translational and rotational diffusion coefcients are given
by StokesEinstein formulas:
D
t
=
k
B
1
6qr
and D
r
=
k
B
1
8q
3
, (9)
where k
B
and T are the Boltzmanns constant and tem-
perature, q and r are the solvent viscosity and radius of
a particle. Depending on size of the particles, time scale of
rotational diffusion may vary signicantly. If we assume
spherical particles with diameters between 1 and 1000nm
ina uid mediumwithlowviscosity, their rotational corre-
lationtimes z
c
=1/(6D
r
) vary inthe range of 10
10
to 10
1
s,
respectively.
Translational self-diffusion can be measured in vari-
ous pulse eld gradient (PFG) experiments [42,43]. Various
theoretical descriptions of the diffusion processes have
been proposed [44]. The theoretical models are based on
different physical concepts such as obstruction effects,
free-volume effects and hydrodynamic interactions.
1.3.5. Concepts of the nuclear Overhauser enhancement
(NOE)
NOE is a manifestation of cross-relaxation between two
nuclear spins which are close to each other in space [45].
It becomes important for the characterization of spatial
relationship between nuclei because it is used for struc-
ture elucidation and conformational analysis. Since NOE
depends on the spatial relationship of two nuclei I and S,
it is a tool for studying intermolecular interactions. In the
classic NOE experiment, the spin I is observed upon per-
turbation of its cross-relaxation partner S, which is usually
achieved by selective irradiation [45]. The NOE is typi-
cally quantied as the fractional change in signal intensity
upon irradiation of S: NOE=[I(perturbed) I
0
]/I
0
, where
I(perturbed) and I
0
are the signal intensity with and with-
out irradiation, respectively.
For the extraction of spatial information the depen-
dency of the NOE on the internuclear distance r
IS
is used:
NOE = exp[(R
1
o
IS
)z
m
][1 exp(2o
IS
z
m
)], (10)
where R
1
represents the total spinlattice relaxation rates
of both the I and S spins, assumed to be equal, o
IS
is the
cross-relaxation rate representing the rate at which NOE is
transferred fromS to I [45], r
IS
is the distance between the
two spins and z
m
is the mixing time.
The study of the early stage of the NOE build-up of the
I enhancement for short mixing times (z
m
0) allows for
determination of o
IS
. Once o
IS
is known, r
IS
can be deter-
mined after evaluation of the correlation time z
c
. However,
themost usedmethodfor internuclear distancedetermina-
tionfromNOEdatarelies ontheuseof acalibrationdistance
[38]. By comparing o
IS
with o
AB
, relative to two nuclei (A
and B) whose internuclear distance (r
AB
) is known, an esti-
mation of r
IS
can be done if the proportionality constant
between o and r
6
is the same for the two pairs of nuclei
(IS and AB):
o
IS
o
AB
=
_
r
IS
r
AB
_
6
(11)
This condition is satised for pairs of nuclei with the
same nature (
I
S
=
A
B
), when the correlation times
of the IS and AB vectors are equivalent. As the effect
decreases with the sixth power of the distance, suitable
distances are limited (usually <5

A).
Measurements of pre-steady-state NOE may be accom-
plished using one- or two-dimensional techniques [29,38].
A 1D technique called truncated driven NOE, or TOE,
involves selectively irradiating the distinct nuclei and
observing the temporal evolution of intensity of the other
peaks. The 2D NOE spectroscopy, NOESY, allows simul-
taneous connectivity of all nuclei in the spectrum to be
established [38]. Instead of saturation, NOESY involves
inversion of signals and observation of the resulting FID.
The NOESY spectrumis represented in a 2Dmap, with each
axis corresponding to a chemical shift scale. The 1D spec-
trumappears on the diagonal, while the off-diagonal peaks
or cross-peaks manifest on close proximity of nuclei.
A number of related experiments appeared recently.
Two are worth mentioning here. The rst of them, DPFGSE-
NOE experiment, is a 1D transient experiment, which
achieves both selective saturation of the target spin and
very cleanremoval of artifacts by the use of gradient pulses.
The second, known as saturation-transfer difference (STD),
involves saturation of the acceptor [46]. Spin diffusion
spreads the saturation over the acceptor and onto any lig-
and that is bound to it. Subsequent exchange of the ligand
into the free-state gives rise to partial saturation of the
free ligand, and thus identies molecules that bind to the
acceptor.
1.3.6. Main sources of NMR signal broadening in gels and
solids
The line width of an NMR signal depends on the internal
mobility of the respective chemical group and the overall
tumbling of polymer molecule. Spectra of isotropic liquids
result in narrowlines, because the molecular motion aver-
ages dipolar couplings of nuclear spins and chemical shift
anisotropy to isotropic values. In contrast, in the systems
like gels, liquid crystals and solids, the motion of certain
segments of the molecules is strongly hindered, thus their
dipolar interactions cannot be averaged out [34], and their
NMR lines becomebroadened. Thelocal magnetic eld, B
loc
,
at a nucleus I generated by a nucleus S is given [39] by:
8
loc
=
S
r
3
IS
(3cos
2
0
IS
1), (12)
where
S
is the magnetic momentum of S, and 0
IS
the
angle between the internuclear vector and the eld B
0
.
The plus and minus signs arise because the spins gener-
ating the local eld may be oriented with and against the
applied eld. If to consider, for example, isolated pairs of
protons in a crystal, one would see two lines with a sepa-
ration which depends on orientation of the proton pairs
in the static magnetic eld B
0
. A polycrystalline sample
produces a spectrum that is superposition of all the pos-
sible orientations of crystallites. In this case, one observes
an assembly average which generates a pattern known as
the Pake doublet [39]. The effect of dipolar interactions in
solid-state spectra is actually more complicated because
we are rarely dealing with isolated pairs of spins. The spec-
trum of abundant spins (like
1
H nuclei) represents broad,
featureless resonances.
The secondmajor source of the signal broadening ingels
is CSA. The electron distribution is not, in general, spherical
or cubic but has a denite directional character resulted in
the directional properties of the chemical shift. Like in the
dipolar interaction, chemical shift depends on (3cos
2
0 1),
where 0 is the angle between the static magnetic eld B
0
and the principal axis of CSA tensor. In a dispersed solid
sample, the small crystals will exhibit a range of orienta-
tions with respect to the applied static eld resulted in a
dispersion of peak positions due to the CSA [39]. In solu-
tion, CSA may produce dramatic inuences on spinlattice
and spinspin relaxation leading to broad lines.
1.3.7. Experimental tools for NMR spectroscopy in
hydro-/organogels
Broadening and/or disappearance of NMR signals in
spectra of the hydro-/organogels often lead to involving
samples where not a gel is present itself, but instead the
gelator is studied in conditions of concentration, solvent,
or temperature that preclude gel formation [47,48]. A key
issue in the study of supramolecular gels by NMR is to dis-
cern if the observed signals correspond to a non-associated
organogelator molecules or oligomeric assemblies. In the
latter case, very valuable information such as intermolec-
ular binding points or the conformation of molecules in
the assemblies could be obtained, for example, by anal-
ysis of chemical shift variations and NOEs, respectively.
Most of the studies on supramolecular gels, dealing with
the variation of chemical shifts with temperature and sol-
vent polarity, as well as those studying NMR relaxation,
implicitly assume that the observed NMR signals corre-
spond to oligomeric molecular assemblies. However, this
behavior is not to be taken for granted for all the gels. For
example, Menger et al. [49] proposed that only discrete
molecules are observable by NMR when a gel of dibenzoyl-
l-cystine was studied, but on the other hand, Duncan
and Whitten [34] concluded that mobile sections of a gel
(therefore associated species) are observable in the studied
system.
In the modern solid-state NMR, it is possible to remove
both the dipolar and CSA broadenings with appropriate
experimental techniques. Lines can be narrowed by the
magic angle spinning (MAS) of the sample with typical
rotational frequencies nowadays of 235kHz [50]. Compo-
nents withalinewidthsmaller thanthespinningfrequency
result in narrow lines. In heterogeneous gels typically a
spectrum as shown in Fig. 4 appears which exhibits both
the narrow lines of mobile components and broad lines of
rigid components.
The experimental NMR techniques allow a structural
analysis of gels when combined with the numeric simula-
tion procedures [37,5154]. The result produces detailed
information on: (i) assignment of every chemical con-
stituent tothe semi-rigidnetwork, or toa liquidcontinuous
phase of gel; (ii) rotational and lateral diffusion of every
constituent; (iii) geometrical parameters of the network
and their changes induced by external inuences; (iv)
phase transitions inthe colloidsystem; (v) morphologyand
porosity of gel; (vi) molecular exchange between phases;
(vii) mechanisms of gel formation and degradation; (viii)
mechanisms and the time scale of releasing the entrapped
substances.
1.3.8. (
1
H)
13
C cross-polarization under MAS conditions
In the cross-polarization (CP) method, a 90
pulse on
1
H
nuclei is followed by a period of simultaneous RF irradi-
ation of
1
H and
13
C nuclei through the contact pulse with
variable duration t
CP
[5254] if the HartmannHahn match
condition [51] is fullled:
1C
= (
C
8
1
) = (
H
8
1
) =
1H
, (13)
where
1
and are the rotating frame precession frequen-
cies and the magnetogyric ratios for proton and carbon.
The
1
H magnetization is larger than the
13
C magnetiza-
tion, so cross-relaxation fromprotons to carbon will cause
the
13
C magnetization to increase by a factor of
H
/
C
.
This factor of almost four is not usually reached in practice
but improves sensitivity because of a strong polarization
of those
13
C nuclei that are subject to dipolar coupling
with
1
H spins. In a standard
13
C CP/MAS experiment with
typical t
CP
=502000s, the signal intensity of carbons
directly bound to hydrogen increases. More rigid systems
result in stronger dipoledipole coupling and, therefore,
more efcient CP transfer. Broadening of CP/MAS NMR
lines can result from several effects, specically changes
in conformation and dynamics. Multiple-pulse line nar-
rowing schemes have an additional feature in that they
remove the
1
H
13
C dipolar but not the scalar spinspin
coupling [50], which contains vast structural information.
The appearance and amplitude of broad (polymer) and
narrow (low-molecular components) lines in the CP/MAS
spectrumof the gel systemdepend on the mixing time t
CP
.
1.3.9. The solid-state
2
H NMR
For the uniformly or selectively deuterated components
of the gel system, the nuclear quadrupolar interaction can
be determined by the C
2
H bond axis orientation relative
to the magnetic eld. Decreasing mobility of the sample
results in the typical Pake pattern [39]. Different types of
molecular motions can be distinguished by observing the
variation in the typical pattern as a function of tempera-
ture. Inthe
2
Hsolid-state NMR spectroscopy the solid-echo
sequence is commonly applied to detect both mobile and
rigid, fast relaxing components [55].
When deuterated water is used as a solvent in hydro-
gel, it can serve as a probe to study the phase equilibrium.
The
2
H NMR spectrumof D
2
O is dominated by the interac-
tion of the deuteron quadrupolar moment with the electric
eld gradients in the nucleus. For an anisotropic sample
the quadrupolar interaction is manifested as a doublet. In
an isotropic solution, on the other hand, this interaction is
averagedto zero as a result of rapidmolecular motions, and
2
H spectrum consists of a sharp singlet. The quadrupolar
splitting^(
2
H), may be expressedinthe context of the con-
ventional two-site model [56], according to which water is
either free or bound. Normally, there is a fast exchange
between the free and bound water on the NMR time scale,
and the ordering of free water molecules is negligible:
^(
2
H) = p
Q
S = n
Q
S
_
X
p
X
w
_
, (14)
wherepis thefractionof bound water,
Q
thequadrupo-
lar coupling constant (220Hz), S the order parameter of
bound water molecules, n the average hydration number
of the polymer, X
p
and X
w
the mole fractions of polymer
and water, respectively.
For a heterogeneous system consisting of two or more
phases, a superposition of the
2
H NMR spectra character-
istic of each phase is expected if water exchange between
phases is slow. However, the quadrupolar splitting itself
does not reveal the structure of the anisotropic phase,
because p fraction in Eq. (14) depends on temperature. To
elucidate the structure, a rotationexperiment, inwhichthe
line shape is measured in dependence on the sample ori-
entation with respect to the external magnetic eld B
0
, has
to be carried out [56].
1.3.10. Relaxation of I =1/2 nuclei in the rotating frame
The transfer of polarization from
1
H to
13
C is controlled
by a rate constant R
CH
, which reects both the magnitude
of the dipolar coupling and motions of the CH bond. In
competition with the build-up polarization of the carbon,
a loss of proton polarization both to the carbon and to the
lattice is inevitable. The loss of magnetization to the lattice
is controlled by a rate constant R
1
, where symbol refers
to the fact that the protons are spin-locked in the frame,
rotating with
1H
=B
1
.
In general, two techniques can be used to monitor
the relaxation process in the rotating frame. In the direct
approach, a 90
pulse on
1
His followed by a spin-lock pulse
(SL) of variable duration t
SL
, where the lateral
1
H magneti-
zation is locked in a transversal orientation. The intensity
of the
1
H signal is detected as a function of t
SL
[50]. Alter-
natively, the cross-polarization sequence can be extended
by a
1
H spin-lock signal with a duration t
SL
between the
initial 90
pulse and the onset of the mixing period. In

this case, the intensity of the
1
H magnetization is detected
indirectly by the amplitude of the resulting (
1
H)
13
C cross-
polarization signal [51]. In the case of a monoexponential
decay of the signal amplitude with t
SL
, the corresponding
timeconstant is assignedtothespinlatticerelaxationtime
of
1
Hnuclei in the rotating frame, T
1
=1/R
1
, which allows
detecting slowdynamic processes.
1.3.11. Magnetic resonance imaging (MRI)
An image delivering MR-measurement consists of two
steps. At rst, the desired effect or contrast (e.g., self-
diffusion or relaxation) will be prepared. In a second step,
the Larmor frequency axis is converted into the space axis
by application of an additional gradient eld G in direction
of the desired prole. For a 3D spatial resolution, it can be
done in each local dimension x, y, and z:
(x, y, z) = [8
0
+C(x, y, z)], (15)
where (x, y, z) is the resonance frequency of the spin at
place (x, y, z) [57]. The spatial resolution ^r depends on the
gradient power and the width of the NMR resonance line
^ of the observed system:
zr =
z
|C(x, y, z)|
(16)
Crosslinked macromolecules show line widths of
0.12.0kHz in dependence on the degree of swelling; sol-
vent molecules of only a few Hz. At a gradient power of
500mT/m this gives a resolution of about 50m, in the
case of restricted diffusion of a fewm.
The self-diffusion can be encoded by the PFG experi-
ment [42,43]. Afurther example of the imaging effect is the
T
2
-contrast of swelling experiments, whichcanbe encoded
by a series of spin-echoes with different echo times. The
contrast roughly scales the microscopic molecular mobility
[57].
Two basic principles of sampling the spatial informa-
tion are used: (i) Fourier imaging (FI) with a Cartesian
sampling of the k-space (k =2/z) and(ii) lteredback pro-
jection (fBP) with a spherical sampling of the k-space. The
advantage of the FI is the simple calculation of the images.
The disadvantage may show up in the higher demands on
the technical realization, including gradient rising times of
only 13s, which are necessary for the measurement of
systems with very short relaxation times, typical of more
solid-like systems. In opposite to the FI, the gradients of
fBP-sequence stay constant during the whole echo pulse
sequence until the next projection angle. However, fBP-
sequences have more difculties in the image calculation.
The ltering process andthe transformationfromspherical
to Cartesian coordinates are very sensitive to artifacts.
2. NMR spectroscopy on the mechanisms of gelation
2.1. Gelation and gelators
Gelation occurs when interconnection between parti-
cles or molecules of the sol increases forcing the sol to
become more viscous thereby losing its uidity. Gel-phase
materials are constructed from high- or low-molecular
weight building blocks that assemble into brillar nanos-
tructures [10,11,58,59]. Despitetheexplosivegrowthinthe
number of structurally diverse gelators (e.g., systems based
on carbohydrates [60,61], peptides and ureas [62], nucle-
obases [63], steroid derivatives [64], dendrimers [65], etc.),
and their high-tech applications, a fundamental under-
standing of the characteristics of molecular self-assembly
and gelation is still, somewhat surprisingly, limited.
The development of useful models to understand
the assembly of molecules into molecular-scale brils
and the mechanism of brilbril interaction to yield
a sample-spanning network is a particularly important
topic [59,6668]. One-dimensional assembly and asso-
ciated network formation underpins the pathology of
neurodegenerative disorders such as Alzheimers disease,
the mechanismof which is sensitive to the precise molecu-
lar structure[69,70]. Recently, afewgroups havedeveloped
advanced models of gelation to explain the transforma-
tion of gelators into three-dimensional sample-spanning
networks.
Aggeli et al. [32] have linked the molecular structure
of a diverse library of peptide molecules to hierarchi-
cal gel formation and developed a self-assembly model
based on the statistical mechanics (Fig. 3). In a key recent
study, Meijer and co-workers [71] considered the self-
assembly of oligo(p-phenylenevinylene) derivatives as a
highly cooperative process involving homogeneous nucle-
ation, initiated by a high-energy non-helical aggregate of
hydrogen-bonded dimers, with solvent molecules play-
ing an explicit role in controlling the process. The similar
cooperativity was detected by self-assembly of bis(urea)
[7276] and pyromellitamide [77] gelators. Terech, Weiss,
and co-workers [78] have demonstrated that gelation
occurs via instantaneous nucleation followed by the one-
dimensional growthof brils. Fages andco-workers [79,80]
suggested that, subsequent to the nucleation and one-
dimensional bril growth, different structural connections
between brils would be an alternative way to modulate
the tertiary gel network, with either gelation or crystal-
lization of the sample being the end result: clearly the
nucleation and growth of gel brils is, at least concep-
tually, related to the crystallization process. Extensive
work by Liu and co-workers [8185] has elucidated a
gelation mechanism based on a supersaturation-driven
crystallographic mismatch branching process. In this
case, a three-dimensional gel network formed when
branching occurred at the tips or on the side faces of
growing brils. McPherson and co-workers [86] showed
that in the two-component gel from bis(2-ethylhexyl)
sodium sulfosuccinate and 4-chlorophenol, the bril for-
mation was based on a nucleation-growth mechanism.
Saha et al. [87] elucidated activation barriers to assem-
bly of riboavin/melamine hydrogels. Remarkable, that
the supramolecular polymerization of pyridyl-terminated
oligomers with trans-dichlorobis(acetonitrile) takes place
in either cooperative or stepwise (isodesmic) manner,
depending on the geometry of the oligomer backbone link-
ages [88].
In summary, for any given self-assembling gelator, it
is highly desirable to understand the formation of large
supramolecular polymeric structures and elucidate the
interactions that mediate their macroscopic organization.
Such studies can potentially generate gelation rules, which
will have a degree of predictive power and thus enable
more advanced design of gelators.
2.2. Sol and gel phases as detected by NMR spectroscopy
The regular high-resolution NMR spectra can be
observed when the material has sol-type properties. For
rigid gel-phase materials, only broad NMR signals (if
any) are observed due to the immobilization of the
low- or high-molecular gelator in the gel network. The
problemcan be resolved when applying solid-state cross-
polarization/magic-angle spinning (CP/MAS), solid-state
Fig. 5. Variable-temperature
1
H NMR spectra of the organogel of all-trans-tri(p-phenilidene vinilidene) (10mg/mL) in benzene-d
6
[92]. Copyright permis-
sion fromAmerican Chemical Society, 2009.
combined rotation and multiple-pulse NMR spectroscopy
(CRAMPS), or swollen-state dipolar decoupled/magic-
angle spinning (DD/MAS) NMR measurements.
Terech et al. [89] studied various organogels made
fromp-octylbenzohydroxamic acid and found that the gel
ber consists of the crystal of the gelator (i.e., dry gel).
The temperature-dependent
1
H NMR measurements for
this dry gel showed that the gelator-originated peaks
completely disappeared in the gel state, while they were
present in the sol state. This feature can be rationalized by
the fact that the gelator molecules almost terminate ther-
mal motion in the crystal. Therefore, disappearance of the
1
HNMR peaks in the gel state seems to be one criterion for
dry gels. However, Sakurai et al. [30] proveda presence of
1
HNMR signals in both the sol and gel states. This suggests
that the gelator molecules still keep the thermal motion in
organogel enough to provide the
1
H NMR spectrum. These
results contrast with those of dry gels. This system was
classied as a wet gel where the solvent molecules are
incorporated into the gel ber.
The above-mentionedtemperature- andconcentration-
dependent changes in the NMR spectra can be applied
for determination of sol-to-gel transition temperature,
T
SG
, [12] as well as the overlap concentration of gela-
tor (or critical concentration of gelation), C*, [90] and for
reconstruction of the whole phase diagrams for hydro-
/organogel systems [91].
Fig. 5 shows the temperature-dependent
1
H NMR
spectra of the organogel of all-trans-tri(p-phenylidene
vinylidene) (TPV) inbenzene, whichprovide aninsight into
TPVself-assembly process [92]. The broadening and subse-
quent disappearance of signals of the aromatic (c, d and
e), O-methylene (f) and vinyl (a and b) protons at 25
C
suggests involvement of strongintermolecular interactions
leading to self-organization of the TPV molecules in the gel
state. The corresponding changes in the aliphatic signals
can also be seen in Fig. 5. Along with gradual increase in
temperature, recovery of the signals was observed, indi-
cating disruption of self-organization due to the thermal
motion of TPV.
As an illustration of the two-component hydrogel self-
assembly [93], Fig. 6a shows the
1
H NMR spectra of
solutions of stearic acid (SA) in chloroform, propanedi-
amine (PDA) in water, and the gel of SA/PDA in water at
various temperatures. For the gel at 25
C, signicant line
broadening of the proton signals of SA was observed. This
suggests that SA was incorporated into the gel, rendering it
solid-like andthus its molecular motionwas restricted. The
signal of the water at 4.78ppmis also broadened, indicat-
ing that the water molecules are part of the gel assembly.
Upon heating the gels, recovery of SA signals was observed
at the gel-melting temperature (45
C).
Similarly, the gel of SA/3,3
-iminobis(propylamine)
(IBPA) inwater showedsignicant line broadening for both
the acids andamines at 25
C(Fig. 6b), thoughuponheating

up to 45
C, the peaks became sharp [93]. This observa-

tion was consistent with the understanding that the SA
molecules experience severe mobility restrictions in a gel,
and that gel melting would indirectly imply melting of the
chains that are now in a state of higher mobility, thereby
averaging out the dipolar interactions between protons.
This fact is proved by the
1
H NMR spectrum of a non-
gel-forming system, that is of myristic acid (MA) and PDA
(Fig. 6c). The signals of neither the acid nor the amines
appear broadened and also no shift of these signals is
observed, indicating isotropic tumbling of the molecules
in solution.
The precursor associates of alkylammonium alkylcar-
bamates forming the chemically reversible organogels in
chloroform were detected by
1
H NMR when gelator con-
centration is raised and temperature reduced [94]. The
associates can serve as the nucleation sites fromwhich the
3D gel assemblies eventually grow in other liquids [32].
At this point, the NMR evidences that the ionic heads of
the ammonium carbamate interact strongly and speci-
cally than their alkyl tails and suggests an inverted micellar
organization. The similar results were obtained in the
course of the detailed multinuclear NMR investigation of
phosphatidylcholine organogels, which were formed after
addition of water to lecithin reverse micelles in cyclohex-
ane [95,96]. Near the gel point, the polar head and the fatty
chains of lecithin show a high mobility, while strong stiff-
ening on the phosphorus atom and adjacent triglyceride
moiety was clearly observable [95].
The sigmoidal shaped dropping in intensity of NMR sig-
nals in the course of gelation (by increase of concentration,
decrease of temperature or variation in composition of the
multicomponent gels) can serve for determining gelation
temperature at the molecular level, T
SG
, which is, normally,
lower than gelation temperature observed visually by the
common vial-inversion method. For example, the fraction
of guanosinehydrazideengagedinthegelationprocess was
determined by
1
H NMR spectroscopy [97]. The difference
between the visually determined gelation temperature
(61
C) and T
SG
(43
C) may be ascribed to the fact that

Fig. 6.
1
H NMR spectra (400MHz) (a): of (A) propanediamine (PDA) in
D
2
O, (B) stearic acid (SA) in CDCl
3
. Hydrogel of SA/PDA (1:3.5) in D
2
O at
(C) 25
C, (D) 35
C, (E) 45
C (gel starts to melt), (F) 55
C (entire gel has

melted). Concentration of SA in hydrogel was 0.07mol/L in each case. (b):
Of (A) 3,3
-iminobis(propylamine) (IBPA) in D
2
O, (B) SA in CDCl
3
. Gels of
SA/IBPA (1:3.5) in D
2
O at (C) 25
C, (D) 35
C, (E) 45
C, (F) 55
C. Concen-
tration of SA in hydrogel was 0.07mol/L in each case. (c): Of PDA in D
2
O
(bottom), myristic acid (MA) in CDCl
3
(middle), and a solution of MA/PDA
(1:3.5) in D
2
O(top). Concentration of MA was 0.07mol/L in each case [93].
Copyright permission fromWiley, 2008.
they concern two different events [72,73]. T
SG
refers to the
variation at the molecular level of the amounts of free and
bound states of gelator, possibly involving motions within
brils without depolymerization. The visually determined
gelation temperature describes the gel-to-sol transition at
the macroscopic level, whenthe material ows under grav-
ity shear because of the cohesion loss. Also, the NMR data
indicate full melting of the gel only at 5560
C [97].
These data allow understanding the relationship between
microscopic and macroscopic collective events in the orga-
nized phases. They also point to the fact that motion within
or exchange in and out of an organized phase may occur
before phase transition.
Yoza et al. [98] measured the
1
H NMR spectra of
1-O-methyl-4,6-O-benzylidene derivatives of d-glucose, d-
galactose, and d-mannose in toluene in the temperature
range of 2583
C. The half-height peak width ^

1/2
of the
PhCHmethine proton is nearly constant above T
SG
, while it
increases with falling temperature below T
SG
. The results
imply that the mobility of gelator molecules is signicantly
suppressed in the gel phase, whereas it is comparable with
that of the homogeneous solution in the sol phase. On
the other hand, plots of chemical shifts of the OH groups
against temperature show that the chemical shifts have
their maximum downeld values at the T
SG
. In general,
the formation of strong hydrogen bonds with OH groups
induces a downeld shift of hydroxyl signals. Hence, the
H-bonds here are strengthened with lowering the temper-
ature. However, H-bonds are gradually weakened as the
temperature falls in the gel phase. Presumably, in the T
SG
region the formation of the soft gel is predominantly
governed by the intermolecular hydrogen bonding, but at
temperatures much lower than T
SG
, the formation of the
crystal-like hard gel [72,73] is governed additionally by
intermolecular interactions, van der Waals interac-
tions, etc. [7476]. When these forces cannot harmonize
with the hydrogen bonding because of steric mismatching,
the H-bonds may be weakened with decreasing tempera-
ture.
In order to evaluate merely the inuence of rheological
properties of the gel system on the
1
H NMR spectrum, it
is necessary to take into account the natural width of the
signals and also the effect of the magnetic eld inhomo-
geneity onthe line width[99]. To this effect, the line widths
of -pyrrolic protons of thegel-formingporphyrinbrucine
conjugate (^
1
) along with those of the non-gel-forming
porphirin-trimethylammoniumbromide (^
2
) were mea-
sured. Since the last one does not form an organogel,
its line width represents the natural line width and the
effect of inhomogeneity of magnetic eld. Thus, the differ-
ence ^^
1/2
=^
2
^
1
reects the contribution of the
gelation process to the line width. Plot of ^^
1/2
vs. tem-
perature showed that the transition occurred at T
SG
=50
C.
A picture grows more complicated in the case of net-
work collapse. Phase transition occurring in three types of
poly(N-isopropyl acrylamide) (PNIPAAm) hydrogels: nor-
mal crosslinked gel (NG), comb-type grafted gel (GG), and
comb-type grafted gel with styrene-modied comb chains
(GG-st), was studied by the variable-temperature solid-
state
1
H HR/MAS NMR [100]. Fig. 7 shows strong decrease
in peak intensities above the so-called lower critical solu-
Fig. 7. Variation in
1
Hpeak intensity of the N-isopropyl group in the PNI-
PAAm crosslinked gel (NG), comb-type grafted gel (GG), and comb-type
grafted gel with styrene-modied comb chains (GG-st) along with a tem-
perature [100]. Copyright permission from American Chemical Society,
2009.
tion temperature (LCST). This decrease was explained by
shrinkingthe swollennetworkintothe tight globule. Inten-
sityof thephenyl peakof thestyrene-modiedcombchains
drops around 30
C by heating that is about 4
C lower than
the temperature at which decrease in the peak intensity of
backbone network occurs. This observation shows that as
temperature increases, the styrene-modied comb chains
collapse rst at a lower temperature (30
C) and then back-

bone polymer networks collapse at a higher temperature
(34
C).
1
H NMR spectroscopy was efciently applied to con-
struct the temperature vs. concentrationphase diagrams of
hydro-/organogels. From the sigmoid variation of the sig-
nal amplitude of methylene groups of 12-hydroxy stearic
acid (HSA) in its nitrobenzene organogel, or the varia-
tion of the ratio of HSA
1
H NMR signal/total NMR area,
the transition temperatures T
SG
were gained to con-
struct the phase diagram [91]. The sigmoid variations
observedduringthe temperature increase (disaggregation)
and the temperature decrease (aggregation) are superim-
posable. NMR transition curve stands slightly above the
curve obtained with the rheometry technique, however
both ways gave the same rheological proles ln() vs.
temperature provided the values ^H
=91.1kJ/mol and
^S
=356.5J/Kmol, consistent with the implication of

strong H-bonds in other gelling systems [11]. In addition,
kinetics of the gelation process can also be extracted from
the time variation of the signal amplitude at a xed tem-
perature, when a liquid solution is put into the gel domain
of the phase diagram. The prole of kinetic curves cor-
responds to a rst-order transition, which is assumed to
be the approximate kinetic result of gelation processes
in molecular gels with a corresponding activation energy
E
a
=24kJ/mol [101].
NMR spectroscopy allows investigation of the specic
properties which are characteristic for polymer hydro-
gels. For example, the stimuli-responsive hydrogels of
crosslinked acrylamide (AAm) based polymers and some
other polymers undergo a collapse or shrinking followed
by phase separation, induced by a small change in exter-
nal parameters like solvent composition, temperature, pH,
etc. The gel volume can decrease 101000 times. On the
molecular level, both the phase separation in solutions
and collapse of crosslinked hydrogels are assumed to be a
macroscopic manifestation of a coil-to-globule transition,
as it was shownfor poly(N-isopropylacrylamide) (PIPAAm)
in water, followed by further aggregation and formation of
stable mesoglobules [15].
NMR investigations of the responsive hydrogels were
perfectly reviewed recently [15]. The broad proton signals
corresponding to polymer segments forming globular-like
structures at temperatures above LCST were detected in
some cases [100107]. For example, for phase separated
and collapsed segments of linear and crosslinked PIPAAm,
line widths of the broad component are about 3kHz [102].
A similar line width 3.7kHz was found also for the
broad
1
H NMR signal of physical gels of linear poly(N,N-
diethylacrylamide) (PDEAAm) [103,104]. Sp ev cek et al.
concluded that the line width 3.7kHz is associated with
isotropic Brownian tumbling of globules as a whole with
the correlation time of 2s. The obtained radius of globu-
lar particles, 15nm, agreed well with the value determined
by small-angle neutron scattering [104].
Ohta et al. [105] obtained
13
C NMR spectra for gelling
PIPAAm, PNIPAAm and poly(methacrylic acid) (PMAAc)
aqueous solutions by using a pulse saturation transfer
(PST)/MAS technique. In this method NOE is used to
enhance the
13
C signals by saturating the proton reso-
nances. They found that chemical shifts of
13
C O and
13
CH
2
carbons decreased and increased, respectively, by
1.5ppm at the phase transition because of the changes
in the number of H-bonds between the carbonyl group
and water. These authors concluded that
13
C CP/MAS NMR
technique is not very efcient because the signals of main
chain carbons disappeared from the spectra both below
and above LCST. On the contrary, Dez-Pe na et al. [106,107]
observed that
13
C CP/MAS NMR spectrum of the same
hydrogel is well-resolved and intense while
13
C PST/MAS
spectrum losses intensity above LCST; only CH
3
group is
mobile enough to produce an intense PST/MAS signal.
13
C NMR allows investigation of the clustering degree
of alkyl groups either in solution or in the gel state.
Indeed, the chemical shifts of the carbon nuclei of the
alkyl groups depend ontheir chemical environment, which
changes whether the hydrophobic tail is free (polar aque-
ous environment) or clustered within brils (low-polarity
environment). If the exchange is slow in the NMR time
scale, a clear peak splitting is observed corresponding to
the free and clustered forms. The splitting of 1625ppm
was detected in
13
C NMR spectra of aqueous solutions
of poly(acrylic acid) (PAAc) modied by alkyl or per-
uoroalkyl side chains [108], as well as allylamine and
dodecylamine side chains [109]. It was estimated by inte-
grating the signals that about 20% of dodecylamine side
chains were clustered in solution and 40% in the gel state.
2.3. Self-assembly of brils and morphology of gels
Gelation starts, normally, with clustering the polymer
chains or low-molecular weight gelators into brils, which
is primarily caused by the association of polar or non-
polar groups, followed by crystallization in the case of
polymers. A good model for self-assembly of polymer b-
rils is poly(vinyl alcohol) (PVA) hydrogel, produced by the
freezethawcycles, the so-called PVA cryogel [110,111].
2.3.1. Self-assembly of hydrogel network frompolymers
and oligomers
Gelation of PVA solutions by freezethaw processing
forms heterogeneous networks withdifferent morphology.
Terao et al. [112] showed that in the
13
C CP/MAS NMR
spectrum of the PVA/water system in the frozen state at
30
C, three peaks I, II and III at 77, 71 and 65ppm for

CH signal, which come from the formation of hydrogen
bonds, appear. It was assigned that peak I comes fromtwo
inter- or intramolecular H-bonds, peak II from one inter-
or intramolecular H-bond, and peak III fromno H-bond. If
the temperature is increased from 30 to 40
C, peak II at
71ppmbecomes broad and the three splitting peaks due to
the triad congurations (mm, mr and rr) appear [113]. This
shows that the molecular motion of PVA chains is much
more decreased, compared with that in the original solu-
tion state due to the formation of hydrogen bonds, and so
CP efciency was enhanced. By repeating the freezethaw
cycles, the fraction of the polymer-rich domains increases,
and then the crosslinked region is formed, which consists
of strong hydrogen bonds.
Willcox et al. [114], using transmission electron
microscopy (TEM) and NMR, detected the formation of
networks with rounded pore morphology, brillar net-
work morphology, or both depending on the number of
freezethawcycles and aging. Formation of crosslinks was
attributed to a kinetically frustrated crystallization in the
rst freeze cycle. The subsequent cycles (or aging pro-
cess) lead to the creation of secondary crystallites and the
growing the primary crystals; however, the mesh spac-
ing slightly changes. With different
13
C NMR experiments
(direct polarization and cross-polarization), they estab-
lished that after the rst freezethaw cycle, the PVA gel
contains a rigid polymer phase of 52%, increasing up to
124% after the 12th cycle.
Analysis of peaks I and II in the
13
C CP/MAS spec-
tra of interpenetrating polymer hydrogels of PVA and
PAAc, prepared by a sequential method (crosslinked
PAAc chains were formed in aqueous solution by
crosslinking copolymerization of acrylic acid and N,N
-
methylenebisacrylamide in the presence of PVA following
by the freezethaw cycles leading to formation of the
PVA cryogel within the synthesized PAAc hydrogel) [115],
allowed detection of PVA/PAAc complex through H-bonds
between the hydroxyl groups of PVA and carbonyl groups
of PAAc.
The multiple-quantum (MQ) NMR spectroscopy is one
of the most versatile androbust quantitative techniques for
investigating not only the structure but also the dynam-
ics of polymer networks [116]. The intrinsic constraints,
irrespective of their nature (crosslinks, entanglements, or
chain packing), lead to non-isotropic segmental uctua-
tions and therefore to the persistence of a weak residual
dipolar coupling, D
res
[117]. The measured effect relies on
the orientation dependence of the uctuating dipolar cou-
pling tensor with respect to the magnetic eld, which can
be described by an orientation autocorrelation function of
the individual chain segments. Fast segmental dynamics in
the range of nano- to microseconds leads to a loss of corre-
lation to a plateau value that is related to the existence of
preferential local orientation generated by the existence of
constraints. In fact, the measurable D
res
is directly propor-
tional to a local dynamic order parameter S of the polymer
backbone[116,118], whichis denedas atimeaverageover
the uctuations of the segment-xed dipolar tensor over
the time until the plateau region is reached.
The MQ measurements indicate that the network
mesh size and the relative amount of non-elastic defects
(i.e., non-crosslinked chains, dangling chains, and loops)
decrease with the number of freezethaw cycles. The for-
mationof thePVAnetworkis accompaniedbyanincreasing
fractionof polymer withfast magnetizationdecay (20s)
[119,120]. The quantitative study of this rigid phase with
a specic refocusing pulse sequence [116] shows that
it is composed of a primary crystalline polymer phase
(5%), which constitutes the main support of the network
structure and determines the mesh size, and a secondary
population of more imperfect crystallites, which increase
the number of elastic chainsegments inthe polymer gel but
do not affect the average network mesh size. Correspond-
ingly, progressivemeltingof thesecondarycrystallites with
increasing temperature does not affect the network mesh
size but only the amount of network defects; and melting
of the main PVA crystallites at 80
C leads to destruc-
tion of the network and formation of an isotropic PVA
solution. Any evidence of formation of covalent crosslinks
between PVA chains during gelation, which were expected
earlier [111], was not detected, because no residual cou-
plings were found after the melting of this primary crystal
fraction.
Effect of compositional microstructure on formation
of crosslinks and mechanism of syneresis was studied
recently in calcium-alginate hydrogels [121]. The
1
H NMR
results obtained from a mixed alginate sample containing
threepolymeric species, guluronateGblocks, mannuronate
M blocks, and alternating MG blocks, without chemical
linkages between the block structures, indicate the for-
mation of mixed junctions between G and MG blocks.
Furthermore, the zipping of long alternating sequences
in secondary MG/MG junctions was suggested to account
for the shrinking (syneresis) of alginate gels in view of its
dependence on the length of the MG blocks.
Peptides and nucleotides are the most promoted
LMWGs to study the ber self-assembly in hydrogels
because application of the advanced NMR techniques
worked out for biomacromolecules [122126]. The gela-
tion abilities of dumbbell-formamphiphilic gelators based
on l-lysine derivatives were investigated [127]. In these
gels, the sol-to-gel transition temperatures, T
SG
, depend on
the carbonnumber of the alkylene spacer. For gelators with
even alkylene spacers, two
1
H NMR signals, corresponding
to the hydrogen-bonded amide protons, were observed in
the sol phase. In contrast, one signal corresponding to the
H-bonded amide protons appeared for gelators with the
odd alkylene spacers. These gelators have two -amide
and two -amide groups as the H-bonded sites. Because
the two -amide and -amide groups in the gelators of
even-numbered chains are antiparallel, it is possible for
the gelators to undergo the four intermolecular H-bonds
through two modes that are between -amide groups and
between -amide groups. In contrast, the amide groups in
the gelators of odd-numbered chains are parallel and they
havefour H-bonds withonebindingmodethat arebetween
-amides and between -amides. The gelators of the even-
numbered chains have strong H-bonds at the center sites
(-amides) and in both sides (-amides), which lead to the
formation of thermally stable gels.
Another group [128] investigated two-component gels
made of a combination of an l-lysine-based dendron
and a rigid diamine spacer (1,4-diaminobenzene or 1,4-
diaminocyclohexane). The molar ratio of dendron/diamine
present in the immobilized gel network was gained with
1
H NMR. Because gelator immobilized within the solid-
like network cannot be observed in the
1
HNMR spectrum
due to line broadening, only material present within the
liquid-like phase had sharp NMR peaks. The mobile gela-
tor can thus be quantied by integration of the peaks
and comparison with an internal standard which does not
associate withthe gel-phase network. Inthis way, the com-
position of the immobile networked gelator bers can be
deduced. It was shown that a dendron/diamine ratio of
1:1 is optimal for satisfying the spatial requirements of
the diamine spacer unit, while promoting intermolecular
H-bonding dendritic head groups.
2.3.2. Self-assembly of LMWG networks
Supramolecular polymers possessing nanoscale super-
structures, such as nanobers and nanoribbons, and helical
structure [129] have gained much attention since they
are capable to form hydro-/organogels [11]. Compared
with common macromolecules, supramolecular polymers,
consisting of arrays of the monomer units linked via
non-covalent interactions, show polymeric properties in
solution and in the bulk and reversible self-assembling
behavior that changes from polymer to monomer with
external stimuli such as temperatures, pH, light, and
electricity. A simplest example of the supramolecular self-
assembly is as follows.
It is knownthat sodiumbis(2-ethylhexyl)sulfosuccinate
(AOT) forms inverse micelles that can incorporate small
amounts of water when dissolved in nonpolar solvents
[130,131]. Tata et al. [132] have reported a transforma-
tion of these inverse micelles to an organogel upon the
addition of suitable phenols to the AOT inverse micelles.
Usually, the optimum gel-forming ratio of AOT/phenol
is unity.
1
H NMR data showed that hydrogen bonding
between the phenolic hydroxyl groups and the surfac-
tant sulfosuccinate head groups lead to the gel formation.
The similar organogels were formed upon the addition
of small quantities of selected resorcinol derivatives 3,5-
dihydroxytoluene and (3,5-dihydroxypentyl)benzene, as
well as 2,6-dihydroxynaphthalene (2,6-DHN) to the AOT
inverse micelles in isooctane [133,134]. The NMR study
of the gel structure reveals signicant restrictions on the
motion of phenols, resorcinols and 2,6-DHN on the NMR
time scale [134]. Based on NMR data, authors supposed
that brils in structure of organogel are formed as the
stacked pile of aromatic rings surrounded by the H-bonded
AOT molecules in the shape of gigantic rod-like micelles
Fig. 8. Schematics of the phenol (resorcinol) gel structure. The phe-
nol molecules are stacked to form a strand with the hydrogen-bonded
AOT adsorbed on the external surface. The lled circles represent AOT
head groups at the back of the strand. The shaded region represents the
hydrophilic shell that envelopes the aromatic ring stack and contains the
phenol hydroxyls and the AOT head groups. The strand could be exible
and contains defects leading to the crosslinks [132b]. Copyright permis-
(Fig. 8). These rod-like micelles resemble large channel-
assembled inclusion complexes formed on the base of
hostguest supramolecular compounds, which are capable
to formsupramolecular networks.
2.3.3. Networks based on the inclusion complexes
It is well known that cyclodextrins (CDs), cyclic starch
oligomers consisting of 6, 7 and 8 -d-glucopyranose
units and named -, - and -CD, respectively, are capa-
ble to incorporate various guest compounds into their
hydrophobic cavities to forminclusion complexes in aque-
ous media [135]. The CD units can gather together by
H-bonds due to the abundant OHgroups in the CDbracelet.
It was reported that mixtures of CDs with polymers pro-
mote supramolecular gel formation which structure was
studied with NMR spectroscopy [136141]. Generally,
free CD molecules retain a less symmetrical conforma-
tion when crystallized in a cage structure with water
as the guest molecules [142]. It is represented by C
1
and C
4
multiple resolved
13
C NMR signals of the glucose
units. Meanwhile, when the polymer chains of grafts are
introduced into the internal cavities of CDs they crystal-
lize in a channel structure and CD molecules acquire a
symmetric conformation, resulting in single C
1
and C
4
res-
onances [136140]. Supramolecular hydrogel formation
based on inclusion complexation in formof polypseudoro-
taxanes between -CD and poly(ethylene glycol)-grafted
chitosan, or poly(-lysine)-grafted dextrans, and -CDand
poly(propylene glycol)-grafted dextrans was investigated
with
13
C CP/MAS NMR. Assembling the similar polypseu-
dorotaxanes was detected with
13
C CP/MAS NMR in PVA
Fig. 9.
1
H ROESY spectra of [N-(2,4,6-trinitrophenyl)-6-amino-trans-
cinnamoyl]--CD in D
2
O at 30
C. The correlations of -CD protons with

the protons of cinnamoyl moiety (a), and the protons of TNB group (b) are
shown [144a]. Copyright permission fromWiley, 2007.
hydrogels with -CD [141]. All of these hydrogels are pH-
and thermo-responsive. Stoichiometry of the - and -
CD/PEG polypseudorotaxane hydrogels was determined
with
1
H NMR; -CD forms polypseudorotaxanes with one,
and -CD with two PEG chains [143].
Chemically responsive supramolecular hydrogels were
constructed by Deng et al. [144] from guest-modied
cyclodextrins. Authors showed with PFG NMR that
self-assembly occurs by formation of supramolecular
LEGO-type brils in an aqueous solution. The 2D
1
HROESY
spectra of -CD/AmTNB and -CD/CiAmTNB (Am=amino,
TNB=trinitrobenzene, Ci =trans-cinnamoyl) reveal that the
TNB and cinnamic groups are included in the cavities of
the neighboring -CD. The strong correlations between the
protons of TNB and the inner protons of -CD were seen in
the spectrum shown in Fig. 9. The H
a
and H
b
protons of
Fig. 10. Supramolecular inclusion complex 1 formed from deoxycholate--CD derivative 2 and azobenzene-branched poly(acrylic acid) copolymer 3
a. Complex 1 can form a hydrogel in aqueous solution, which has a gelsol phase transition induced by light-responsive trans-cis isomerization of the
azobenzene units. b
1
H ROESY spectrum(500MHz) of complex [147]. Copyright permission fromAmerican Chemical Society, 2009.
the cinnamoyl moiety show strong correlations with the
protons H
3
and H
5
of -CD, respectively. Furthermore, the
protons of the TNB parts showcorrelations with H
5
and H
6
of -CD, which is indicative of a tail-to-head instead of a
tail-to-tail structure.
Inclusion complexes were detected by NMR in
thermo-responsive hydrogels prepared by polymerization
of hydroxypropyl--CD (HP--CD)/glycidyl methacrylate
(GMA) complex and N-isopropylacrylamide (NIPAAm)
[145]. For HP--CD, the chemical shifts of H
3
, H
5
, H
6
, and
methyl protons which are located inside or close to the rim
were examined. All the corresponding signals were shifted
upeld in the presence of the guest GMA. These shifts are
indicative of the inclusioncomplex. Simultaneously, for the
guest molecule GMA, C
1
, C
2
, C
4
, C
5
, C
6
, and methyl carbons
showed downeld
13
C shifts, while C
3
shifted in the oppo-
site direction. This observationsuggests that all the protons
of GMA are located inside the cavity of CD except of C
3
and
its protons. Integration of the
1
H NMR signals of GMA and
HP--CDsuggests that the epoxide group is located within
the cavity of a CD and the carbonyl group within the cavity
of another CD.
Atwo-stage precipitation polymerization in an aqueous
solution was used to prepare -CD/PNIPAAm coreshell
thermo-responsive microgels [146]. At the rst stage, core
hydrogel particles containing CD moieties were synthe-
sized by copolymerization of NIPAAm with a monovinyl
-CD monomer. At the second stage, using the core par-
ticles as seeds, PNIPAAm shell was added by NIPAAm
polymerization. According to the peak intensities in the
1
H
NMR spectra of microgels, the actual molar ratio of -CD
component to NIPAAmunits in samples was calculated to
be 1:100 or 2:100. This result evidenced a presence of -CD
moieties within the core.
In the fascinating work of Stoddart and Zhao [147],
a deoxycholic acid-modied -CD derivative 2 and an
azobenzene-branched PAAc copolymer 3 were prepared
(Fig. 10a), and the association/dissociation of 2 with the
trans/cis-azobenzene units in 3 were characterized by UV
spectroscopy, ICD, and
1
H ROESY NMR. The experimental
data indicated that the trans-azobenzene units are bound
strongly within the cavities of 2 whereas cis-azobenzene is
not bound at all. A supramolecular inclusion complex 1,
formed by 2 and 3, is accompanied by the formation of
a hydrogel. The light-responsive gel-to-sol and sol-to-gel
phase transitions are induced by trans-cis photoisomer-
ization of the azobenzene units. In the hydrogel system,
the trans-azobenzene units in 3 are included inside the
hydrophobic cavity of 2. UponUVirradiationof 355nm, the
hydrogel is converted into the sol phase because the trans-
azobenzene units are converted photochemically to their
cis congurations, whereupontheresultingcis-azobenzene
units dissociate from2. The hydrogel canbe recoveredfrom
the sol by photoirradiation with visible light of 450nm.
The
1
H ROESY spectrum of 2 displays cross-peaks
between the H
3
and H
5
protons on the inside of the -
CD bracelet and the key protons of the deoxycholate unit.
This suggests that the deoxycholate unit in 2 is either self-
included in the hydrophobic cavity of -CD to form the
intramolecular inclusioncomplexor includedinanother -
CD bracelet to form the intermolecular inclusion complex
(Fig. 10a) [147]. Thefact that theazobenzeneunit of copoly-
mer 3 can be included in the cavity of deoxycholate--CD
(2) was demonstrated by another
1
H ROESY experiment
(Fig. 10b). The cross-peaks between H
of azobenzene and
H
3
of the -CD ring (peak A), H
and H
3
(H
5
) (peak B), and
H
and H
5
(peak C) were detected. However, there were
no cross-peaks found between H
3
and H
5
of the -CD ring
and the protons of the deoxycholate unit in 2. These obser-
vations indicate that the trans-azobenzene unit is included
in the hydrophobic cavity from the secondary face of the
-CD as illustrated in Fig. 10a, and the deoxycholate unit
is excluded from the hydrophobic cavity. This structural
information cannot be gained fromany other experiment.
It is well known [33] that the steroid units can participate
in 1D stacking aggregation, driven by intermolecular van
der Waals and/or hydrogen bonding interactions. Since the
deoxycholate unit in 2 is included into the cavity of -CD,
this may well prevent thedeoxycholateunit in2frominter-
molecular association to form the hydrogels. Inclusion of
the trans-azobenzene unit in the cavity of deoxycholate--
CD (2) orients the deoxycholate unit away fromthe cavity,
and the free deoxycholate units in complex 1 can interact
with each other, thus favoring the formation of the hydro-
gel.
Poly(oxyethylene) (PEO) containing a 4,4
-
diaminodiphenylmethane (DADP) moiety with thiol
groups at the terminals and its inclusion complexes with
- and -CDs were prepared [148]. The complexation-
induced shifts in
1
H NMR spectra were observed at the
aromatic protons of DADP, when mixing CDs and DADP
solutions in water. The upeld chemical shift of signals of
H
3
, H
5
, and H
6
of - and -CD exceeded that of H
1
, H
2
,
and H
4
. This means that the H
3
and H
5
atoms are directed
toward the interior of the cavity of - and -CD, whereas
the H
1
, H
2
, and H
4
atoms are located on the exterior. These
results indicate that DADP molecules are located within
cavities of - and -CD. For -CD and DADP, the changes
in ^y for the aromatic protons of DADP on the Jobs plot
were plotted as a function of the mole fraction of DADP
to give a peak at 0.67, indicating the formation of a 1:2
complex. For -CD and DADP, the changes in ^y for the
aromatic protons of DADP gave a peak at 0.50, indicating
the formation of a 1:1 complex. Each molecule of these
inclusion complexes can be consistently exchanging each
other. Therefore, it was reasonable concluded that the
junctions of -CD including DADP moieties can act as
mobile crosslinks.
Any other types of cavitands like, for example, biscal-
ixarenes [149] and crown ethers [150], which can form
inclusion complexes, may be used to construct responsive
hydro-/organogels as well. For example, theself-assembled
nanostructures were prepared with the CO
2
gas, which
employ both H-bonding and thermally reversible carba-
mate bonds [149]. As precursors, calixarene ureas were
synthesized, which strongly aggregate/dimerize in apo-
lar solvent with the formation of self-assembled capsules
and linear polymer chains, respectively, and also possess
CO
2
-philic primaryaminogroups ontheperiphery.
13
CO
2
effectively reacts with these polymer chains and crosslinks
themwiththe formationof multiple carbamate salt bridges
of the 3Dpolymer network. This supramolecular organogel
with the
13
C-labeled crosslinks was characterized by
13
C
NMR spectroscopy [149]. Addition of a competitive solvent
destroyed H-bonds in gel, but did not affect the carbamate
linkers. On the other hand, thermal release of CO
2
from
organogel was easily accomplished. Thus, the 3D polymer
network was transformed back to linear polymeric chain
without breaking up. This system opens indeed the way
for switchable materials, which reversibly trap, store, and
then release guest molecules.
The remarkable stabilization of the sugar-based crown
ether hydrogel in the presence of alkyl diammonium ions
as guests was found based on the
1
HNMR titration [150]. It
was shown that the hostguest type interaction stabilizes
such hydrogel by synergic effects of H-bonds.
2.3.4. Stereochemical control of the self-assembly
Molecular packing in the gel phase is signicantly
affected by conguration and conformation of LMWG
[19,151162]. This inuence can be detected only with the
aid of NMR spectroscopy. For example, the carbohydrate
amphiphiles are packed within organogel in a head-to-
tail fashion [151]. If there is, however, the possibility
to form interlayer H-bonds, as in the case of N-n-octyl-
d-gluconamide or N-n-octyl-d-gluconamide-6-(3-pyridyl
carboxylate), the molecules are packed head-to-head.
Some gluconamides, e.g., those with aliphatic substituents,
express their molecular chirality in the supramolecular
structures, whereas others, in particular those containing
a large aromatic substituent on carbon atomC
6
, yield non-
chiral aggregates, probablyduetointerferingstacking
of the substituents.
The conguration control of ber self-assembly
through the photoinduced gelation was shown for oxa-
lyl amide derivatives bearing 4-dodecyloxy-stilbene
and bis(PheOH)-maleic acid and fumaric acid amides
[158160]. The trans derivatives acted as versatile gelators
of various organic solvents, whereas the corresponding
cis derivatives showed poor gelation ability or none at all
(similar to system in Ref. [147]).
1
H NMR spectra demon-
strated that the gelation process occurred because of a
rapid cis trans photoisomerization followed by a self-
assembly of the trans molecules. The dissolution enthalpy
values ^H
obtained for trans-stilbene derivatives were

61 and 57kJ/mol in toluene and DMSO organogels, respec-
tively. The same was observed by
13
C CP/MAS NMR and
variable-temperature
1
HNMR measurements; the ordered
trans-conformation of alkyl chains of N
-octadecyl-N
-(4-
vinyl)-benzoyl-l-phenylalanine amide plays a crucial role
in the molecular packing in cyclohexane organogel which
is dominated by van der Waals interactions [152].
Photo-responsive supramolecular hydrogels were dis-
covered based on rational design coupled with a combi-
natorial library of glycolipids [153].
1
H NMR spectroscopy
and various microscopy techniques showed for the opti-
mal hydrogel that the trans-cis photoisomerization of the
double bond in the fumaric amide unit effectively caused
assembly or disassembly of bers to yield the hydrogel or
the corresponding sol. The
1
H NMR studies of the sol indi-
cated that 89% of the fumaric (trans) formwas converted to
the maleic (cis) form. The gel-to-sol transitionis dependent
onthe trans/cis ratio, anda thresholdcontent of at least 50%
of the cis formis needed for this transition.
Gelator chirality has a pronounced inuence on the
gelation properties [19,155157,161,162]. It was shown
by
1
H NMR that, in most cases, racemates are less
efcient gelators than pure enatiomers [155,156]. The
chiral bilayer effect was formulated to explain why
pure enatiomers prefer to gel, while racemates of
amphiphilic gelators prefer to crystallize [157].
1
H NMR,
FTIR and X-ray diffraction studies of four chiral bis(amino
alcohol)oxalamides (14: amino alcohol =leucinol, vali-
nol, phenylglycinol, and phenylalaninol, respectively) as
LMWGs revealed that the primary organization motif
of (S,S)-1 and racemate 1 (rac-1) in lipophilic solvents
forminverse bilayers [155,156]. Within these bilayers, the
gelator molecules are connected by cooperative H-bonds
between oxalamide units and OH groups, while the inter-
bilayer interactions are realizedthroughlipophilic contacts
between the iBu groups of leucinol. Oxalamide meso-1
lacks any gelation ability and crystallizes in monolayers. In
dichloromethane rac-1 forms an unstable gel; this is prone
to crystallization as a result of the formation of symmet-
rical meso bilayers. In contrast, in aromatic solvents rac-1
forms stable gels consisting fromenantiomeric bilayers. In
the 1D NOESY experiments, positive NOEs, which conrm
the presence of non-aggregated gelator molecules, were
observed. However, negative NOEs were obtained for the
gel sample. Negative NOEs are knowntoappear inthe spec-
tra of macromolecules with a molecular mass in excess of
1500 [72,73]. Therefore, their occurrence in the gel spec-
trum clearly shows that aggregates of at least six gelator
molecules are present in the entrapped toluene [156].
To acquire further information on the gelber orga-
nization, the temperature-dependent NMR spectra of
solutions of (S,S)-1 and rac-1 in toluene were recorded. For
the NH, C*H, and methylene (CH
2
OH) groups, two chem-
ical shift trends can be observed, namely downeld shifts
in the 4060
C range, and upeld shifts in the 6090
C
range. Disintegration of the oxalamideoxalamide and
OHOH H-bonds explains the upeld shifts for NH
and the methylene protons at higher temperatures. The
C*H protons oriented parallel to the oxalamide carbonyl
groups are also shifted upeld at higher temperatures,
presumably, because of destruction of the H-bonds. The
methylene(C*CH
2
), methine, andmethyl signals of theiBu
groups were shifted downeld when the temperature was
increased [19]. This suggests the presence of van der Waals
interactions between the iBu groups in the aggregates.
Hence, the NMR data for both (S,S)-1 and rac-1 suggest
that the ber organization is characterized by intermolec-
ular hydrogen bonding of the oxalamides and OH groups,
and by van der Waals interactions that involve iBu groups
[155,156].
The synergic gelation effect in the two-component
gels was reported [161]. An equimolar mixture of
(S,S)-bis(LeuOH) oxalamide, (S,S)-1, and (S,S)-bis(leucinol)
oxalamide, (S,S)-2, is able to gel up to 7 times larger a vol-
ume of p-xylene than an equal mass of each component
and up to 5 times larger a volume than an equal mass of
the (S,S)-1+(R,R)-2 or (S,S)-1+rac-2 equimolar mixtures.
The homochiral (S,S)-1+(S,S)-2 combination of gelators is
capable of hardening a volumes up to 5 times larger of
certain solvents than the heterochiral (S,S)-1+(R,R)-2 com-
bination.
In the
1
H NMR spectrum of the (S,S)-1+rac-2 toluene
gel, the (S,S)-2- and(R,R)-2-NHs are clearly non-equivalent,
appearing at 7.89 and 7.70ppm, respectively. Heating
the gel sample leads to disaggregation of the assem-
blies and the two signals of the non-equivalent rac-2 NHs
merge into a single one, showing that upon disaggre-
gation the NHs have become equivalent. These results
clearly show that the non-equivalency of the (S,S)-2 and
(R,R)-2 NHs must be a consequence of their interac-
tion with (S,S)-1, resulting in diasteroisomerism at the
supramolecular level [161]. Hence, different morphologies
and temperature-dissolution proles observed for (S,S)-
1+(S,S)-2 and (S,S)-1+(R,R)-2 toluene gels is the result
of diastereomeric assemblies existing in these systems
at the certain level of supramolecular organization. The
obtained results suggest on the specic solvation proper-
ties of gelled solvent and its specic interactions with gel
assemblies, which in certain cases (toluene and m- and
p-xylene) result in different solvation of diastereomeric
assemblies andleads tothe different gel morphology, while
in other solvents (benzene and o-xylene) both diastere-
omeric assemblies are similarly solvated, which results
in the similar morphology [162]. Hence, each component
tends to formreversed bilayers inlipophilic solvents which
theninteract and organize into gel bers. The bers formed
by interaction of the (S,S)-1 and (S,S)-2 bilayers and of the
(S,S)-1 and (R,R)-2 bilayers are diastereoisomeric. In cer-
tain solvents, such diastereomeric bers give different gel
morphologies andgellingefciencies. This observationwas
denoted as the synergic gelation effect [161].
2.3.5. Thermodynamics of gelation
Based on
1
H NMR data, the equilibrium constant
values K
d
can be calculated, and the cumulative disso-
lution enthalpy ^H
and entropy ^S
changes can be
obtained either for one-component [58,154156], or two-
component gels [163]. In these investigations species are
denedas soluble if they are visible, andas insoluble aggre-
gates if they are not visible by NMR [58]. If, however,
the assembly is alternatively isodesmic (with a continu-
ous distribution of monomer/oligomers), there is a poorly
dened transition frommonomer to extended aggregates,
and the NMR treatment is much more complex. In the non-
isodesmic case, the newly added material is incorporated
into the NMR silent solid-like gel network. For an ideal
solution, the solubility (Sol) at a given temperature can be
expressed by the vant Hoff equation:
ln(Sol) =
zH
R1
eq
+
zS
R
, (17)
where T
eq
is the equilibrium temperature and R is the
gas constant. A typical vant Hoff plot of ln(Sol) vs. 1/T
can be used to calculate ^H
and ^S
, and furthermore,
extrapolation of these data permits the solubility at differ-
ent temperatures to be determined. Speva cek pioneered
in applying the vant Hoff equation for the analysis of
NMR data obtained for s-PMMA gels [154]. Measurements
are, normally, made at concentrations well above C* and
temperatures below T
SG
, in order to avoid experimental
conditions relating to the initial stages of gelation.
For example, for (S,S)-1 and rac-1, the ^H
values
obtained at 25
C were 93 and 78kJ/mol, while the ^S
values were determined as 240 and 197J/(mol K) [155],

respectively. It appears that both dissolution processes are
slightly enthalpy controlled. Hence, below T
SG
, the differ-
ence in solubility and thermodynamic parameters reects
the difference in the gel network ber dimensions.
Rizkov et al. [163] showed that the dinitrobenzoate
moieties conjugated to cholesterol can assemble two-
component donoracceptor organogels with as much as
a 1516-fold larger amount of polyaromatic hydrocar-
bons (C-18 alkyl chain). Based on
1
H NMR data authors
calculated K
d
values and thermodynamic parameters of
gelation for two types of the yellowand white gels: ^H
=
207kJ/mol, ^G
=68kJ/mol, ^S
=465J/(mol K), and

^H
=229kJ/mol, ^G
=78kJ/mol, ^S
=507J/(mol K),
respectively. In this case, the entropy change constitutes
the major driving force for dissolution.
Hirst et al. [58] highlighted the key role of solubility in
gelation process and established that T
SG
and C* depend
on solubility. By employing a vant Hoff analysis of solu-
bility data, obtained from
1
H NMR measurements, authors
acquired thermodynamic parameters, followed by the cal-
culated temperature T
calc
for complete solubilization of the
networked gelator. The concentration dependence of T
calc
allowed at the rst time to rationalize plateau-region
thermal stability values in terms of gelator molecular
design. This was demonstrated for four gelators with lysine
units attachedto eachendof analiphatic diamine, withdif-
ferent peripheral groups (Z or Boc) in different locations on
theperipheryof themolecule. Bytuningtheperipheral pro-
tecting groups of the gelators, the solubility of the system
is modied, which, in turn, controls the saturation point of
the systemand, hence, the concentrationat whichnetwork
formation takes place. In Table 1 is shown that the critical
concentration of gelator incorporated into the solid-phase
network, C
crit
, is invariant of gelator structure. However,
because some systems have higher solubility, they are less
effective gelators and require the higher total concentra-
tions to achieve gelation, hence shedding light on the role
of the C* parameter.
A two-stage phase transition process was detected by
NMR in the course of gelation of the crosslinked PNIPAAm
in the presence of NaCl and CaCl
2
aqueous solutions [164].
Thermodynamic quantities wereobtainedfroma vant Hoff
analysis of the temperature-dependent K
d
values, which
were derived from the
1
H NMR data. The ^H
and ^S
values for the polymer hydrogel in water are 3.4kJ/mol

and 11.2J/(mol K) for stage I, which was attributed to
the hydrophobic interactions between neighboring iso-
propyl groups. The formation of hydrogen bonds during
stage II yielded ^H
and ^S
values of 14.8kJ/mol and

48.4J/(mol K). However, the corresponding ^H
values
in 150mM NaCl or CaCl
2
are reduced to 1.5 and 1.8kJ/mol
for stage I of the dehydration process. This corresponds
to the known effect of salts on the hydrophobic interac-
tion energetics. The value of ^S
also decreased to 4.9 and

5.9J/(mol K) in NaCl and CaCl
2
solutions, respectively.
However, the thermodynamic parameters during stage II
were only slightly affected by the salts. The lower tem-
peratures required to induce spontaneous precipitation
imply that ^G
of precipitationis reduced. The equilibrium

thermodynamics shows that salts have a great effect on
hydrophobic interactions associated with the phase tran-
sition process and on the stability of PNIPAAm(and other)
hydrogels.
2.4. Hydration and hydrogen bonding in gels
Thewater-polymer/-ber interactioninhydrogels is the
subject of many NMR studies [15,46,105107,165180].
The particular relevance of this method is that it has
provided a means for independent analysis of the water
by using
1
H,
2
H,
17
O, and of the polymer chains/LMWGs
with
1
H,
13
C, and
15
N nuclei. The information that could,
thus, be derived concerns both the molecular dynamics
(from relaxation times) and hydrogen bonding (from the
induced chemical shifts). The ability of water to form
hydrogen bonds with the polymer chains and hence com-
pete withpolymerpolymer H-bondentanglements allows
an increase in polymer mobility and mobile chain confor-
mations.
Two groups of investigators showed with
1
H and
13
C
NMR that dehydration, or syneresis, is a principal factor
of the sol-to-gel transition for polymer gelators [165167].
For example, the signals at 72.272.6ppmin the
13
C NMR
spectra of poly(Ala) (PA) or poly(L-Ala-co-L-Phe) (PAF) end-
capped with poly(propylene glycol)-poly(ethylene glycol)-
poly(propylene glycol) (poloxamer or PLX), PAPLXPA or
PAFPLXPAF, in aqueous solutions showed a broadening
as the temperature increased from 20 to 40
C in which
the sol-to-gel transitionof the PAPLXPAor PAFPLXPAF
aqueous solutions occurred. The broadening of the NMR
peak is an indication of a decrease in the molecular motion
of the PLX that has been claimed for the dehydration of the
block. Hence, it was concluded that the dehydration of the
PLX, as well as the conformational change in polypeptide
from random coils to -sheets, plays a critical role in the
sol-to-gel transition [166,167].
Water is often termed bound if it has reduced mobil-
ity and slightly different structure as compared to neat
or free water. Andrade and Jhon [168] reported that
considerable interactions occurred between the water
molecules and the polymer networks by the reduced value
Table 1
Thermodynamic and solubility parameters obtained from
1
HNMR data and thermal parameters of gelation for one-component gelators designed to exhibit
structural variations of - and -amino groups of l-lysine coupled to diaminononane [58].
Gelator
a
^H
, kJ/mol ^S
, J/(mol K) Solubility, mM T
SG
d
T
calc
e
C* at 25
C, mM C
crit
f
at C*, mM
At 40mM
4-Boc 45 119 31
b
30 35 25 4
2-Z 101 286 3
b
45 51 10 6
2-Z 103 259 0.3
b
80 86 3 3
4-Z 106 252 0.01
c
88 111 3 3
a
4-Boc four groups of tert-butyl carbamate, 2-Z and 4-Z two or four groups of the benzyl carbamate substituents.
b
Calculated directly fromthe
1
H NMR measurements at 30
C.
c
Calculated fromthe extrapolation of vant Hoff plot at 30
C.
d
Obtained fromthe simple gel tube inversion method.
e
The calculated temperature for complete solubilization of networked gelator, based on the vant Hoff treatment of solubility data.
f
Concentration of gelator present in the brillar network at C*, derived from
1
H NMR.
of the spinlattice relaxation time,
1
H T
1
, of the imbibed
water (e.g., 0.132s for poly(2-hydroxyethyl methacrylate),
PHEMA, containing 50wt.% of water) over that of neat
water (4.50s at 307K). An increase in the total water con-
tent resulted in an increase in the fraction of free water
and a decrease in the fraction of bound water. Addi-
tionally, when the water content of PHEMA lies below
25wt.%, the values of the spinspin relaxation time,
1
H T
2
,
are constant (5ms) and rapidly rise as the water content
increases (up to 35ms at a water content of 50wt.%), sup-
porting the proposed three-step imbibition process [169].
McBrierty et al. [170173] reported that the trend of T
1
, T
1
and T
2
as a function of temperature for hydrated PHEMA
points toward complex water behavior. The minima in the
plots of T
1
and T
1
as a function of temperature at 225 and
200K, respectively, and the appearance of a long compo-
nent in T
2
at 180K were interpreted as the onset of motion
in the glasslike water. In addition, a hysteresis effect was
observed in T
1
and the long component of T
2
between 240
and 280K.
Advanced solid-state
1
H NMR was applied for study-
ing the H-bond formation occurring in polymer hydrogels
based on PNIPAAm, PMAAc and their poly(NIPAAm-
co-MAAc) copolymers [106,107]. The nature of the
pH-dependent collapse of PMAAc hydrogels was estab-
lished. In aqueous solution, PMAAc changes from an
expanded conformation at high pH to a compact con-
tracted format lowpH, where H-bonds play a central role.
In
1
H MAS spectra, dried PMAAc samples previously col-
lapsed at lowpH have characteristic signals in the spectral
region of the carboxylic acid protons. With the aid of 2D
1
H double-quantum (DQ) MAS NMR spectra, three sig-
nals were distinguished at 8, 10.5 and 12.5ppm, which
were attributed to free carboxylic groups and two differ-
ent types of H-bonded forms, respectively. The 12.5ppm
signal arises from the H-bond with the shortest HH
distance, corresponding to the most stable form. The
weaker H-bonded form (at 10.5ppm) requires a slightly
lower pH, while the free acid signal (at 8ppm) emerges
under the most acidic medium. The stabilities of the H-
bonded carboxylic acid dimers can be inferred from the
protonproton distances within the dimers, i.e., 275 and
295pmfor the protons at 12.5 and 10.5ppm, respectively,
which were determined by means of DQ MAS sideband
patterns.
Two populations of water were distinguished in
1
H
MAS spectra of copolymers, one of which is probably
situated near stable H-bonded regions, while the other
behaves similarly to the free water [106,107]. For collapsed
copolymers and interpenetrating polymer networks, the
appearance of characteristic signals shows that H-bonding
occurs betweenNIPAAmandMMAc units. Thetemperature
dependence of the DQ spectra indicates that acid-amide
H-bonds formed between both co-monomers are more
stable than the acidacid H-bonds formed among MAAc
moieties alone. Finally, the pHdependence of the DQspec-
tra demonstrates that hydrogen bonding phenomena are
directly related to the polymer collapse.
Solid-state
2
H NMR qualitatively demonstrated the
presence of two types of water within hydrogel of poly(dl-
lactide-co-glycolide)/D
2
O: bulk water with free rotation
(narrow signal) and bound water with hindered rota-
tion (broad signal) [174]. The quantitative measurements
showed that the polymer contained a constant amount of
bound water.
The bound water can be efciently detected with mag-
netization transfer (MT), or z-spectroscopy [175180].
MT proles of hydrogels are obtained from the offset-
frequency dependence of the intensity of the water signal.
An off-resonance pulse, applied to saturate the resonances
of the solid protons of polymers, indirectly affects the
magnetizationof thewater, becauseduringthepulsecross-
relaxation through dipolar coupling occurs between the
protons of the bound water and those of the polymer
[175,176]. When the spectrum of water is acquired after
theoff-resonancepulse, theintensityof signal will decrease
relative to the intensity in the absence of the preparation
pulse, reecting the fast exchange process and the amount
of cross-relaxation that has occurred. The MT-spectrumis
the plot of the normalized signal intensity, M/M
0
, vs. the
offset of the presaturation pulse. Fig. 11 shows the MT-
spectra for some of the hydroxypropyl methyl cellulose
(HPMC) hydrogels. Only water nuclei in close proxim-
ity to the polymer, i.e., bound water, can participate in
cross-relaxation with the polymer protons because the
magnitude of the dipolar coupling interaction is inversely
proportional to the internuclear distance cubed. The MT-
spectrum of distilled water is included to show the result
of the same experiment on a sample where the possibil-
ity for cross-relaxation is absent. The width-at-half-height
Fig. 11. Magnetizationtransfer NMR spectra for selectedHPMChydrogels
from observation of the water signal. The mixtures shown are distilled
water (solid line), 9.4% (led circles), 14.2% (empty circles), 18.7% (lled
squares), 38.3% (empty squares), and 56.6% (triangles) of HPMC. A prepa-
ration pulse with an excitation width of 500Hz was applied at different
offset frequencies for 5s prior to the acquisition of the water signal. Only
the range of offsets of 30 to +30kHz is shown [175]. Copyright permis-
of the MT-spectrum is related to the width of the spectral
line for the solid protons, even where the spectrumof the
solid component is not observed directly. The less mobile
are the polymer chains, the broader are the lines for the
polymer protons and the corresponding MT-spectrum for
the water in the polymer. Fig. 11 demonstrates that the
widths of the MT-spectra increase with increasing polymer
concentration.
The MT NMR was used to probe the effects of con-
centration, degree of hydrolysis, and temperature on the
formation of a network in aqueous solutions and gels of
atactic PVA [177]. Because, for example, the MT proles of
the PEO solutions do not broaden as viscosity increases,
the change in prole area of the PVA solutions and gels
may be ascribed with condence to polymer association
andconsequent network formation. Ingeneral, the spectral
lineshape of a sample with a single motional component is
described by a Lorentzian function. The MT proles of aged
PVA samples cannot be described by a single Lorentzian
function, because they contain an additional component
with a short
1
H T
2
due to immobilized polymer. These MT
proles were t to a function that is the sum of a narrow
Lorentzian and a broad Gaussian components. The mobile
component is the part of the systemcontaining both poly-
mer and water that gives rise to the Lorentzian line shape,
while the immobile component is the part containing both
polymer and water with collectively restricted motion that
gives rise to a Gaussian line shape. A sample with an MT
prole that is 50% Gaussian or greater is more solid-like
than liquid-like. As a result such a sample may be consid-
ered a gel fromthe point of viewof NMR.
Magnetization transfer occurs both directly via dipo-
lar interactionbetweenwater andpolymer/LMWGprotons
and indirectly via proton exchange between labile poly-
mer/LMWG protons and water, with the rst mechanism
being more relevant for the carboxyl groups in the
anionic form and the second being predominant for the
protonated amine groups [178180]. It was unclear, how-
ever, how water is affected by the gelators and whether
specic functional groups within the gelators are respon-
sible. To address this aspect, Mahajan et al. [46] utilized
saturation-transfer difference (STD) NMR experiments,
wherein the water was irradiated with a saturating pulse,
andthe subsequent transfer of magnetizationtothe gelator
(-glutamyl-cysteinyl-glycine (GSH)-pyrene) protons was
monitored, to detect the site-specic interactions between
water and gelator within the hydrogels. With this strategy
it is possible to compare STD spectra in the solution state,
in a sample that is just beginning to gel, and of the gel state,
using the same sample.
To generate the STD spectrum, rst the water signal is
pre-irradiated for a sufciently long time to achieve satu-
ration. Next, a control spectrum is recorded without any
presaturation on the water signal. The difference of these
spectra yields an STD spectrum. The intensity of any indi-
vidual proton signal in the STD spectrum depends on the
off-rate of water fromthat site, the saturation time, and the
proton concentration in the gel. Water molecules tightly
boundtospecic protons cantransfer magnetizationonthe
NMRtimescale. However, if thegelator protons interact too
strongly with the water, the magnetization will be trans-
ferred only from the small subset of water that is bound
to it. More efcient saturation transfer is obtained for the
entire population if the off-rate of water from the proton
of interest is fast relative to the saturation time, so that
manywater protons caninteract withgelator, but alsoslow
enough to allowtransfer of magnetization.
The STD amplication factor was calculated for peaks
of the GSH group, pyrenyl protons, and the amide pro-
tons of the GSHpyrene gel [46]. The amplication factors
cluster into groups dened by the type of proton. The
aliphatic GSH protons have the highest amplication fac-
tors in both solution and gel states. The pyrenyl protons
have near zero amplicationfactors insolutionbut become
spread over a wide range including some with factors
as high as 30%, in the gels. For the single amide peak
observed in both states, the amplication factor actu-
ally decreases upon gelation. Therefore, STD NMR studies
demonstrate that water interacts strongly with GSH-borne
protons in both solution and gel states, but only the gels
include waterpyrenyl interactions with signicant resi-
dencetimes [46]. Theclose-packed pyrenes donot interact
with solvent as effectively, and they are relatively rigid
compared to the more mobile GSH moieties, which lie on
the outside of the bers and have access to solvent. How-
ever, although the interaction of water with the pyrenyl
groups is less strong, the formation of gel coincides with
an increase in the pyrenylwater interactions, which are
essentially absent in the solution state. A critical demon-
stration of the STDresults is that the hydrophobic moieties
are not excluded frominteractions with water in the gels.
Rather, the pyrene groups specically participate in sol-
vent interactions in the gel state, presumably through
cooperative interactions with the GSH groups. These STD
experiments suggested parallels between hydrogelation
andproteinsolvation[181], whereinhydrophobic aromatic
groups can interact strongly with water when nearby polar
residues can also participate, and this is likely to be the
critical feature of gelators.
Intermolecular H-bonds, the common driving force
for aggregation of gelators [11,16], lose their strength in
water unless many are combined in a cooperative manner
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0
a b
c
d
Ha
Hc
Hd
Hb
sol
N
H
N
H
N
H
N
H
O O
gel
/ ppm
Fig. 12.
1
H NMR spectra of 1-methyl-2,4-bis(N
-octadecaneureido)-
benzene (MBOB) solution in DMSO-d
6
at 110
C (sol) and organogel at

30
C (gel) [182]. Copyright permission fromElsevier, 2008.

and protect from solvent. Instead, hydrophobic interac-
tion which lacks the precise directing ability of H-bonds
becomes the most important force in aqueous environ-
ments [21]. NMR data in the gel and sol phases allow
indicating the extent of H-bonding in the aggregate or ber
precursor, normally, by shifting NMR signals of the protons
which are capable to form H-bonds toward the high eld
in the course of gelation [16,92,93,127,132,154156,158
160,182210]. Though, investigator has always to remem-
ber that study of gel precursors withthe NMR spectroscopy
can demonstrate only ability of formation of H-bonds (or
stackings, see Section 2.5), but not an actual presence
of these in hydro-/organogel itself.
The typical
1
H NMR spectra of 1-methyl-2,4-bis(N
-
octadecaneureido)benzene in DMSOsolution at 110
C and
the organogel at 30
C are shown in Fig. 12 [182]. The sig-

nals of four NHgroups are shifted from7.85, 7.62, 6.08 and
5.73ppmin solution to 8.19, 7.69, 6.43 and 5.90ppmin the
gel state, respectively. These shifts to lower eld indicate
that the four NH groups are involved in the formation of
H-bonds in the course of the sol-to-gel phase transition.
Thetemperatureandconcentration-dependent
1
HNMR
spectra of N,N
-oxalyl-bis(ValOH)/CH
3
CN/CHCl
3
organogel
reveal involvement of oxalyl amide and carboxylic groups
in intermolecular hydrogen bonding [155,156]. The upeld
shifts of oxalyl amide signals upon temperature increase
correspond to dissociation of small LMWG assemblies. In
the concentration-dependent spectra the NH signals are
shifted downeld until the critical gelation concentra-
tion is reached. Thus, at the pre-gelation concentrations
the assemblies are formed by intermolecular H-bonding
between oxalyl amides.
A self-associating tripeptide Boc-Ala(1)-Aib(2)--
Ala(3)-OMe (where Aib=-amino-isobutyric acid) [183]
and some other tripeptides [184] form thermoreversible
gels in various organic solvents. Concentration-dependent
1
H NMR experiments were carried out in order to investi-
gate which NH groups are involved in the intermolecular
H-bonding. The results reveal that a change in the chemical
shift for NH(3) is only 0.21, whereas for NH(1) and NH(2)
these changes are 0.45 and 0.38ppm, respectively. The
temperature dependence of the chemical shifts for the
gelator in benzene has been measured in the tempera-
ture range from 25 (gel state) to 70
C (sol state). Again,

signicant changes in chemical shifts have been observed
for NH(1) and NH(2). However, the shift for NH(3) does
not alter appreciably. This indicates that NH(1) and NH(2)
are involved in intermolecular H-bonding and that NH(3)
may form a weak intermolecular H-bond, apart from its
participation in intramolecular H-bonding.
Intermolecular H-bonding of the amide NH and the
indole NH was found to be one of the regulating factors for
the self-assembly of the l-tryptophan-based amphiphiles
[185,186]. The amide NH shifted upeld (from 8.55 to
7.79ppm) as the water content increased up to 30%, and
then moved downeld (to 7.99ppm) as the amount of
water increased further. In contrast, the indole NHshowed
a continuous upeld shift (from11.09 to 10.29ppm). Upon
initiation of the self-assembly process, the bulky indole
group possibly twists toward the hydrophobic domain of
the aggregate, thereby exposing the carbonyl group in the
aqueous phase. Thus, conformational changes presumably
forced the amide NH to move toward the hydrophobic
region, resulting in upeld shift of both the NH signals.
Above 30% of water content, intermolecular H-bonding
might occur between the amide NH and the carbonyl
oxygen, which deshields the amide NH. Moreover, as the
water content increases, the ammoniumheadgroup of the
molecule becomes hydrated, leading to the upeld shift
of the neighboring protons of amide NH and indole NH.
Above 30% of water content, the amide proton possibly
starts participating in the intermolecular H-bonding, lead-
ing to dehydration and ber formation and resulting in
the downeld shift of amide NH. In case of indole NH, the
upeld shift continued, due presumably to the increasing
interactionof theparallel indolemoieties, as well as H-
bonding with water. In the gel state at 30
C, a broad peak
was observed in the aromatic region, following by split-
ting with a downeld shift (from 7.22 to 8.08ppm) as the
temperature increased from30 to 80
C. The aromatic pro-

tons were shielded, due possibly to the interaction
of the indole rings in the gel structure. As the tempera-
ture increases, the intermolecular H-bonds are disrupted,
leading to the transition fromgel to sol. The rise in temper-
ature also eliminates the hydrophobic stacking of indole
moieties, resulting in the downeld shift of the aromatic
signals.
More sophisticated temperature dependence was dis-
covered in
1
H NMR spectra of LMWGs based on L-Lys with
alkylpyridinium or -imidazolium groups in DMSO/H
2
O
mixtures [187190]. It is clearly seen in Fig. 13 that along
with increasing water content, signals of the amide pro-
tons shift to lower eld for up to 30% additions. Above this
level, they thenshift upeld. These changes inthe chemical
shifts reveal that the H-bonding with DMSO (>S OHN)
replaces that with H
2
O (H
2
OHN), and then with inter-
molecular H-bonding between the amide groups. On the
other hand, the signal of the pyridiniumprotonH
c
is shifted
to the opposite direction: upeld up to a 30% H
2
O con-
tent, and then into lower eld. The addition of water brings
about the hydration of the charged pyridinium segments.
This leads to the upeld shift. On further addition of water,
gelator molecules self-assemble into the brils and dehy-
dration probably takes place.
Fig. 13.
1
HNMR spectra of N
-(11-pyridiniumundecanoyl)-N
-lauroyl-l-
lysine ethyl ester bromide in DMSO-d
6
containing various ratios of water
[188]. Copyright permission fromWiley, 2003.
In order to give an evidence of H-bonding as a driv-
ing force by formation of gel bers, McPherson and
co-workers [191] examined the
1
H spectra of both the
organogel components, AOT and p-chlorophenol. When p-
chlorophenol is dissolved in isooctane in the absence of
AOT, the peak corresponding to the hydroxyl proton is
broadened due to H-bonding between the p-chlorophenol
molecules and is not visible. As soon as AOT is added to
the p-chlorophenol solution in isooctane, the peak corre-
sponding to the hydroxyl proton becomes visible, and the
difference between the chemical shifts of the o- and m-
protons of p-chlorophenol is reduced. This indicates that
p-chlorophenol molecules prefer to H-bond with AOT at
very low p-chlorophenol concentrations, even before any
clusters are evident in the system. There is no dramatic
change in the spectra for p-chlorophenol when clusters
become observable. But on transformation into the gel,
the peaks corresponding to the o- and m-protons of p-
chlorophenol undergo signicant line broadening. This
suggests that the p-chlorophenol molecules that were
labile on the NMR time scale when in clusters become
rigid upon the formation of a 3D network [132]. When
a trace amount of acetone is added to a rigid gel, it
breaks down into a sol, and the peak corresponding to the
hydroxyl proton reemerges. The gel breaks down as ace-
tone molecules compete with AOT molecules in forming
H-bonds with p-chlorophenol.
The rst organogel system in toluene which does not
transfer to sol in the temperature range of 2580
C was
described by

Zini c and co-workers [192]. The chiral gela-
tors are constructed from11-aminoundecanoic acid, lauric
and the core peptide unit. The temperature changes in pro-
ton chemical shifts, as well as disruption of intermolecular
H-bonds by addition of DMSO, which is the strong com-
petitor for hydrogen bonds, to toluene clearly showed that
the intermolecular H-bonds involve amide and carboxyl
groups.
Formation of cooperative H-bonds was detected in
1
H NMR spectra of poly(acrylamide-co-acrylic acid),
poly(AAm-co-AAc), hydrogels [193]. During the formation
and dissociation of H-bonds the two hydrogen nuclei of
amide group experience different chemical environments.
The chemical shifts of 7.69, 7.63 and 6.91ppm corre-
spond to the hydrogen nuclei in NH
2
OH
2
, NH
2
NH
2
and
free NH
2
, respectively. With the increase of AAc content
in copolymers, the double peaks at low eld gradually
approach each other and become one peak. The forma-
tion of H-bond between hydrogen of amide group in PAAm
and oxygen of carboxyl group in PAAc makes the signals
shift downeld as compared to the H-bonds among amide
groups of PAAm. ThesecooperativeH-bonds areconsidered
the main driving force by formation of thermosensitive
hydrogels.
Variable-temperature
1
H and
31
P NMR was applied to
monitor the gelationprocess of chitosan/glycerophosphate
(CS/GP) thermosensitive hydrogel [194]. Investigations
showed that
1
H signals of CS moved upeld, but
31
P signal
of GP moved downeld along with increase of tempera-
ture. In addition, the
1
Hchemical shifts in CS had a turning
point aroundthegelationtemperature, indicatingdramatic
change in the chemical environment. The gelation mecha-
nism of CS/GP hydrogel was deduced as follows: with the
increase of temperature, electrostatic interaction between
CS and GP is broken down, followed by formation of H-
bonds between CS chains, which lead to forming brils.
Formation of intermolecular H-bonds of the cholesteryl
glycinate ferrocenoyl amide (CGF) molecules was sup-
ported by
1
H NMR measurements of the gel of CGF/ethyl
acetate [195]. The signal of the NH group was shifted
upeld from 7.15 to 6.98ppm with increasing tempera-
ture, indicating anincrease inthe strengthof the NHbond,
direct evidence of the disentanglement of NHfroma bond-
ing state. The concentration-dependent shift of NH signal
also supports the involvement of the group to formation of
gel network. A similar result was reported for gelation of
binaphtalene derivatives in chloroform[196].
The dimeric cholesteryl-based A(LS)
2
LMWGs, which
consist of an aromatic group (A) connected to a steroidal
moiety (S) through a linker (L), diacid amides of
dicholesteryl l-phenylanilate [197199] together with
glucose-contained cholesteryl [200], were studied. The
temperature and concentration dependencies for chemical
shifts of amide signals in
1
H NMR spectra shown in Fig. 14
reveal that H-bonding between the gelator molecules is an
important driving force for the gel formation. As usually,
the signals of the amide protons were shifted gradually to
a lower eld with increasing the concentration of LMWG
and with decreasing of temperature. On the contrary, the
Fig. 14.
1
H NMR spectra of the dimeric cholesteryl-based gelator of A(LS)
2
in CDCl
3
: (a) temperature dependence at a concentration of 4wt.%; (b)
concentration dependence at 298K [197]. Copyright permission fromWiley, 2008.
aromatic signals shifted upeld with increasing the LMWG
concentration testify on possible stacking of benzene
rings, which also contributes to the gelation [197]. Spec-
tra of a compound with the phthaloyl linker, which did not
formorganogel at all, did not showany shifts for aromatic
signals, what indicates no stacking in this particular
case.
The very similar conclusion on H-bonding as a main
driving force of the gelation process was made after
study of concentration and temperature dependencies
of chemical shifts in the
1
H NMR spectra of dioc-
tadecyl l-glutamic acid derivatives [201], 2-benzoyloxi-
and 2-benzoylamino-5-cyanotroponoids and their cor-
responding benzenoids [202], pyrene-containing glucose
derivative [203], cyclo(L-Tyr-L-Lys) andits 3-aminoderiva-
tives [204] and oligophenylethynyl conjugates bearing
long-chain pyridine-2,6-dicarboxamides [205].
Dendrimers, given their multiplicity of functional
groups and hence their potential for forming multivalent
supramolecular interactions in a network-type structure,
have been of great interest for the formation of gel mate-
rials. Love et al. [206] employed variable-temperature
1
H NMR studies to investigate the nature of the pep-
tidic dendrimerdendrimer interactions. The NMR spectra
were observed for toluene solutions of the l-lysine-based
dendritic gelators. For rigid organogels, only broad NMR
peaks were observed. In the case of G1-SS-G1 (G means
generation of a dendrimers, SS disulde bridge), peaks
were observed in the
1
H NMR spectrum, indicating some
mobility of the individual dendrimers across the entire
temperature range. The upeld shift of the NH peak
of carbamate from 5.90 to 5.77ppm by increasing the
temperature from 20 to 50
C indicated that H-bonds

involving this NH group are being broken on heating.
For G2-SS-G2, no signal was observed at 20
C, indi-
cating the dendrimers are immobilized within the gel
network. However, on heating from 50 to 80
C, the NH
signals appear and shift upeld, indicative that hydro-
gen bonds involving these NH protons are being broken.
For G3-SS-G3, the spectra essentially remained broad and
featureless up to 80
C, implying immobilization of the

dendrimer molecules, but they appear at 80
C which cor-
responds to H-bonded amide and carbamate NH peaks.
Based on these data, it was concluded that the gel net-
workis largelyheldtogether byH-bonds betweendendritic
headgroups of individual gelator molecules. This pro-
vides an enthalpic stabilization of the network which is
reected in the dendritically elevated T
SG
values. The sim-
ilar conclusions based on
1
H NMR data were revealed for
dendrons based on glycine and l-glutamic acid from the
rst generation (G1) to the third generation (G3) [207],
as well as for butylamide-terminated poly(amidoamine)
[208] and the dumbbell-shaped dendritic molecules with
p-terphenylene core (Gn-TP-Gn) [209].
It was mentioned yet, that NMR study of H-bonding
of gelator molecules allows making the denite conclu-
sions on structure and molecular packing of brils as the
main structure elements of gels. Yabuuchi et al. [210]
showed based on
1
H NMR data that two urea groups in
pyridine-based bisurea gelator form different H-bonding
patterns. Only small shifts were observed for the signal of
NH adjacent to the alkyl chain in the pyridine-based gela-
tor, while the signals of NH adjacent to the pyridyl unit
showed downeld shifts as the concentration increased.
These results indicate that the urea NH adjacent to the
alkyl chain is involved in intramolecular H-bonds in the
gelator molecules. This hydrogen-bonded structure in the
self-assembled bers is shown in Fig. 15a, where one of
two urea units is involved in intramolecular H-bonding
with the pyridyl nitrogen, while another urea unit forms
bifurcated intermolecular H-bonds. It is a key structure
for the brous self-assembly along with the efcient gela-
tion because in the case of benzene-based bisurea gelator,
which showed poor gelation ability, only bifurcated inter-
molecular H-bonding (Fig. 15b) is possible.
2.5. stacking and van der Waals interactions
The spontaneous self-assembly of amphiphilic com-
pounds is aconsequenceof thesynergyof anumber of weak
Fig. 15. Hydrogen-bonded structures of pyridine-based (a) and benzene-
based (b) bisurea gelators [210]. Copyright permission fromRSC, 2003.
forces in a delicate balance [21]. Small molecules in which
the relationship between a rigid aromatic and hydrophobic
core and its surrounding exible coils, which can behave as
hydrophobic or hydrophilic units, are outstanding exam-
ples of amphiphilic systems capable to assemble into a
variety of well-ordered non-covalent crosslinks [16]. For-
mation of these superstructures is often dominated by
interactions of the aromatic backbone surrounded by
a variable number or hydrophilic chains [211]. However,
interactions, considered as the sum of several non-
covalent forces like electrostatic, attractive and repulsive
orbital interactions and solvophobic effects [212214], are
not the onlysupramolecular forces participating inthe self-
assembly and some other non-covalent forces cooperate in
the formation of the supramolecular ensembles [215].
One of the simplest organogel systems self-assembled
from the amphiphiles is one of the above-mentioned
that is formed upon the addition of small quantities of
dihydroxynaphthalenes to the AOT inverse micelles in
isooctane [132134]. Hydrogen bonding between AOT and
the dihydroxynaphthalenes is responsible for positioning
the dihydroxynaphthalenes as bridges between micelles
followed by their stacking like it is showninFig. 8. The
proposed microstructure of the gel is deduced from three
key observations: (i) the low concentrations of selected
dihydroxynaphthalenes that indicate minimal perturba-
tions of the AOT micellar structure, (ii) the
1
H NMR
spectroscopic results that reveal the selective motional
rigidity of the dopant dihydroxynaphthalene species, and
(iii) the necessity of hydroxyl groups on opposite ends
of the dihydroxynaphthalenes for the gelation. Such a
comprehensive approach to prove the existence of
stacking as a driving force of gelation is quite appropriate.
Unfortunately, therearealot of papers, wheretheproposed
models of molecular packing in hydro-/organogels based
allegedly on the NMR data, remain the consistent men-
tal pictures. Therefore, we will discuss further only those
works where the thorough experimental studies were per-
formed in order to justify the proposed models of gel
microstructure.
The results obtained by two groups of investigators
[155,156,216218] are the rst direct NMR evidences for
the stacking inthe gel phase, andthey suggest that the
amphiphilic gelators can form well-ordered rigid bilayer
structures inwater throughstacking of phenyl groups,
H-bonding and hydrophobic interactions in the gel state.
Zinic et al. [155,156] showed that
1
H chemical shifts
in the NMR spectra of bis(PhgOH)/D
2
O/DMSO (3:1) gel
revealed deshielding effects with temperature increase
(20
C, gel,
Ph
=7.24 and
C*H
=5.13ppm; 80
C, solution,
Ph
=7.27,
C*H
=5.26ppm), which can be explained by dis-
sociation of aromatic stacked assemblies dissolved in the
entrapped solvent and being in equilibrium with the gel
network. For bis(LeuOH), duetosimilar gelationproperties,
it was assumed that the favorable lipophilic interactions
and hydrophobic effects between isobutyl groups could
stabilize self-assembly of bers in water (Fig. 16). Because
intermolecular hydrogen bonding is highly disfavored in
water due to strong competitive solvent effects, H-bonds
can form in water if sufciently large primary associates
appear enabling formation of H-bonds in the co-operative
manner. Consequently, the gelation of water can be ratio-
nalized by the initial formation of elongated primary
assemblies driven by intermolecular lipophilic and
stacking interactions. Such assemblies may further orga-
nize into brils or bril bundles by lateral H-bonding
involving both the oxalyl amide and carboxyl groups
(Fig. 16).
The similar results were obtained by Jung et al.
[216218]. They showed that aromatic peaks in
1
H NMR
spectrumof amphiphile, dodecanoyl-p-aminophenyl--d-
aldopyranoside gel (1) appeared at 7.43 (H
b
) and 7.38ppm
(H
a
) at 27
C. Upon heating, the newpeaks appeared grad-

ually at 7.60 (H
b
) and 7.28ppm (H
a
) with disappearance
Fig. 16. Schematic presentation of bis(PhgOH) (Phg denotes R-phenyl
glycine) and bis(LeuOH) self-assembly in hydrogel bers based on phenyl
stacking, dispersive andhydrophobic interactions. Thick lines denote oxa-
lyl amide fragments laying in the plane A perpendicular to the plane of
drawing. Intermolecular H-bonding between self-complementary oxalyl
amide units in plane A and lateral H-bonding between carboxylic groups
is indicated [155]. Copyright permission fromWiley, 2001.
of the original peaks. The difference in chemical shifts
between the aromatic signals H
a
and H
b
may arise from
stacking and the H-bond interactions if the induction
effect of the hydrogen bonding is too large to cancel the
upeld shifts of H
a
due to stacking. The appearance of
theseparatedsignals of theaggregatedandnon-aggregated
species demonstrates that the chemical exchange is slow
onthe NMR time scale. Onthe other hand, original aromatic
proton signals (6.77 and 6.58ppm) of the aqueous bolaam-
phiphiles, 1,12-dodecanedicarboxylic-bis(p-aminophenyl-
-l-aldopyranoside)s gel (2), displayed gradual downeld
shift (7.27 and 6.97ppm) when heated, but gave no new
signals as with aqueous gel (1). This behavior is due to
the fact that the chemical exchange between the self-
assembled and monomeric species is fast on the NMR
timescale. The bolaamphiphilic gelators can form mono-
layer structures through stacking between phenyl
groups in the gel state. The similar downeld shift was
detected for all the aromatic protons in hydrogel from
N-(4-pyridyl)isonicotinamide while heating and disrupt-
ing stacking between pyridyl groups [219]. The NH
peaks of the organogel (2) were gradually shifted upeld
from 8.97 to 8.61ppm upon heating, an indication that
the amide group of the bolaamphiphilic gelators partic-
ipates in the intermolecular H-bond interaction in the
gel state. The similar picture was observed recently for
one-component organogels of N-(4-n-alkyloxybenzoyl)-l-
alanine amphiphiles [211] and two-component hydrogels
of melamine and gallic acid. [220].
Garcia et al. [221,222] investigated self-assembly of the
good model gelator which provides many opportunities
for stacking of the aromatic units, rectangu-
lar oligo(phenylene ethynylene) amphiphile, by using
concentration-dependent and variable-temperature
1
H
NMR experiments. As the concentration increased, a slight
shielding of most protons was observed. This shift pattern,
in which all the signals are affected upon increasing con-
centration, clearlyindicates that the radial oligo(phenylene
ethynylene) systems are arranged in an eclipsed fash-
ion. The self-assembly is induced by a face-to-face
stacking of the aromatic units reinforced by the van der
Waals contacts between the peripheral triethylene glycol
chains. Broadening and slight shielding of signals fromthe
triethylene glycol chains was observed upon decreasing
temperature. The concentration and temperature depen-
dencies of the chemical shifts showed a linear behavior,
although with different slope values for the aromatic or
aliphatic signals. This observationwas attributedto the dif-
ferent nature and participation degree of the non-covalent
forces involved in the interaction of aromatic fragments or
triethylene glycol chains.
It was shown that some other good LMWGs for realiza-
tion solely stacking in the structure of organogels are
4
-(N,N-diphenylamino)biphenyl-4-yl/aniline [223],
a dicationic platinum(II) terpyridyl complex, [(t-
Bu3tpy)Pt(OXD)Pt-(t-Bu3tpy)](PF
6
)
2
(t-Bu3tpy=4,4
,4-
tritert-butyl-2,2
:6
,2-terpyridyl, OXD=2,5-bis(4-
ethynylphenyl)[1,3,4]oxadiazole)/acetonitrile [224],
T-shaped asymmetric bisphenazine containing cyano-
phenyl groups in methylene chloride [225] and periph-
erally dimethyl isophtalate-functionalized poly(benzyl
ether) dendrons [18]. These gelators do not contain any
conventional H-bonding moieties, but consist only from
aromatic rings.
In the
1
H NMR spectrum of 4
-(N,N-
diphenylamino)biphenyl-4-yl, a line-broadening effect
arising from stacking was observed in the gel sample
at 20
C. At the temperature elevated up to 70
C, the
broad peaks became sharp according to the gel-to-sol
phase transition [223]. The
1
H NMR spectra of dicationic
platinum(II) terpyridyl complex, showed that when the
temperature of its CD
3
CN solution was decreased from
50 to 20
C, the signal for the pyridyl protons H

6
, H
3
,
H
3
and H
5
on the terpyridyl ligand shifted upeld from
9.14, 8.36, 8.33, and 7.79ppm to 8.94, 8.15, 8.17, and
7.62ppm, respectively. However, the signal for phenyl
H
a
on the OXD moiety shifted downeld from 8.12 to
8.19ppm and the signal corresponding to H
b
remained
constant. The ratio of the intensities of aromatic signal and
the solvent decreased at lower temperatures. A dramatic
broadening of all the signals was observed below 0
C,
which is consistent with complete gel formation. Lu et al.
[224] identied intermolecular interactions between
the [(t-Bu3tpy)PtC C] moieties as the main driving force
for gelation.
Jang et al. [225] have demonstrated the utility of the
cyanophenyl group in T-shaped asymmetric bisphenazine
containing a large and at aromatic core to induce 1D
assembly and manipulate assembling morphology. As the
concentration increased, the signals of all aromatic protons
shifted upeld by a range of 0.120.33ppm. The upeld
shift of aromatic protons and the aliphatic protons nearby
the aromatic core is due to the shielding effects of the
aromatic ring above the respective protons and indicative
intermolecular stacking. Besides, the self-association
constant of each proton, K
a
, was estimated from these
data. The proton of H
c
had the highest K
a
of 30.2L/mol
among others. Though, the K
a
value is still lower than that
for structurally similar pyrazine-containing fused aromatic
compounds without t-butyl groups [226], implying that
extensive stacking was suppressed by bulky t-butyl
groups. As a result, two different driving forces, stack-
ing and cyano-interaction competitively induced different
assembled structures depending on the concentration.
Feng et al. [18] reported on a peripherally dimethyl
isophtalate-functionalized poly(benzyl ether) dendrons,
which contain no conventional gelating motifs but form
gels in various aromatic and polar organic solvents, and
even in aqueous media. The stackings of dendrons in
solution and the gel state were conrmed by
1
HNMR stud-
ies. Insolutions of dendronG1inCDCl
3
/CCl
4
, theincreasein
the concentration resulted in the usual slight upeld shifts
of the signals for the aromatic protons of the peripheral
dimethyl isophtalate and internal benzyl rings. Regard-
ing the temperature-dependent experiments, these signals
were found to be gradually shifted downeld when the
temperature increased from10 to 50
C. Notably, the posi-

tion of signals corresponding to the benzyl ring at the focal
point remainedconstant. Asimilar shift trendwas observed
upon warming of dendron G2 in CD
3
CN from 20 to 90
C.
In addition to the shifts, these signals experienced a dra-
matic broadening below60
C, which is consistent with the

complete gel formation.
Therefore, H-bonding, stacking and van der Waals
interactions are the main driving forces of gelation,
which provide formation of the non-covalent or phys-
ical crosslinks between bers self-assembled from the
LMWGs in supramolecular hydro-/organogels. Between
other driving forces in physical gels we can mention also
electrostatic attraction [11], solvatophobic [10,214] and
salting [227] effects, which have been still studied with the
NMR spectroscopy only at the limitedlevel. Another type of
crosslinking, formationof the chemical crosslinks is mainly
characteristic for the polymer gelators.
2.6. Chemical crosslinking
Methods of NMR spectroscopy allow detecting the
chemical crosslinks, formed in the course of gelation, as
well as estimation of the crosslinking degree, an important
structural parameter that affects the swelling and mechan-
ical properties of a gel. To this effect, normally,
1
HHR/MAS
and
13
C CP/MAS are applied. This structural information is
unique from the quantitative point of view and comple-
mentary to the data which could be obtained with FTIR
and Raman spectroscopies. In many cases studies were
limitedonlybyidenticationof crosslinks [228237], how-
ever, in the more comprehensive investigations, a degree
of crosslinking was determined as well [238253].
2.6.1. Model studies of the initial stage of crosslinking
A few successful attempts to apply the high-resolution
1
H,
13
C and
31
P NMR spectroscopy without MAS to study
the initial stages of crosslinking in polysaccharide hydro-
gels were done for hyaluronan (HA) crosslinked with
1,3-diaminopropane (1,3-DAP), 1,6-diaminohexane (1,6-
DAE) [244] or tyramine [250], and -d-glucopyranoside
(-d-Glc-OMe) as a low-molecular model compound
for pullulan crosslinked with sodium trimetaphosphate
(STMP) [245].
The chemical modication of HA was focused on
its two principal functional groups: hydroxyl and car-
boxyl.
1
H NMR spectroscopy was used to characterize
tyramine substituted hyaluronan (TS-HA) as a result of
the carbodiimide-mediated reaction [250]. In the spec-
trum of tyramine, the signals for both pairs of aromatic
ring protons (6.97.3ppm) and aliphatic side chain pro-
tons (2.93.3ppm) were identied. Besides, there are
two additional aromatic signals at lower eld as com-
pared to the spectrum of tyramine alone. These signals
result from the formation of the tyramine-O-1-ethyl-3-
(3-dimethyl aminopropyl) carbodiimide (EDC) adducts.
Hence, these results conrmthe presence of both tyramine
and tyramine-O-EDC on TS-HA.
In the carbonyl region of
13
C NMR spectrum of the
crosslinked HA, the lower eld signal was assigned to the
carbonyl of the N-acetyl glucosamine unit, which is spe-
cic to the non-crosslinked HA, and the higher eld signal
was assigned to the carbonyl of the glucuronic unit (GlcA).
The signal at 170ppm was assigned to the carboxylate
condensed with the diamine. The signals at 38ppm can
be attributed to the carbons C
1
and C
3
, whereas the sig-
nals at 25ppm to the carbons C
2
of the crosslinked
and non-crosslinked 1,3-DAP, respectively. The degree of
crosslinking was obtained by comparing the carbonyl sig-
nals of the crosslinked and non-crosslinked GlcA [244].
The
1
H,
13
Cand
31
PNMR studyof thecrosslinkingmech-
anism with STMP in polysaccharides was reported using
-d-Glc-OMe as a model [245]. It was shown that the rst
step reaction of STMP with Glc-OMe gives grafted sodium
tripolyphosphate (STPPg). Onthe one hand, STTPgcanreact
with the second alcohol functionality to give a crosslinked
monophosphate. On the other hand, a grafted phosphate
could be obtained by alkaline degradation of STPPg. The
31
P
31
P and
1
H
31
P COSY spectra allowed obtaining the
crosslinking density of STMPpolysaccharide hydrogels.
In order to performthe similar model investigations of
adducts appearinginthecourseof crosslinkingthepolysac-
charides themselves, the high-resolution MAS technique
is necessary. For example,
1
H HR/MAS NMR was applied
to establish xanthan gum derivatives obtained by reac-
tion with acrylic acid or acid-reactive derivatives (acryloyl
chloride, maleic anhydride), which take part in the devel-
opment of 3D hydrogel network [251]. The presence of
acrylate and maleate groups in the modied structure of
xanthan gum was detected. The degree of substitution as
determined by
1
H NMR could be controlled by varying the
chemical nature of crosslinking agent, reaction time and
temperature.
2.6.2. Estimation of the crosslinking degree
The incorporation of the adipate and pendant six-
carbon hydrazido-linkers was veried by
13
C CP/MAS on
lyophilized samples of hyaluronic acid [238]. The spec-
trum of derivatized HA resembled that of HA itself, with
an additional intensity of signals fromthe carboxyl groups
of the pendant moiety and in the aliphatic region between
25 and 35ppm. The intensity in the aliphatic region had
the characteristic behavior of a methylene carbon in the
delay-without-decoupling experiment. The signals at 34
and 25ppm corresponded to the calculated values for
adipate - and -carbons, respectively. After subtraction
of the two spectra of crosslinked and non-crosslinked
HA, the residual intensity in the aliphatic region was
deduced. It corresponded to that expected for functional-
ization of 17.521.5% of the glucuronate carboxyl groups of
HA. For the hydrazido-HA, integration of non-overlapped
crosslinker peaks in the unsubtracted spectra allowed esti-
mation of the efciency of crosslinking, which was in the
range of 1025% fromthe available glucuronate moieties.
In contrast to the hydrazido-HA, the signals for
the crosslinked HA hydrogels showed clear evidence of
conformational changes in the HA backbone [238]. In HA-
3,3
-dithiobis(sulfosuccinimidyl propionate), the major

13
C
peak was shifted upeld by 3.5ppm. This was evident as
side-by-side negative and positive peaks in the difference
spectrum, suggesting that the natural abundance sugar
backbone signals have been shifted upeld. These changes
were alsodetectedinthe other crosslinkedderivatives, e.g.,
in HA-bis(sulfosuccinimidyl)suberate.
1
H HR/MAS NMR was successfully applied for estima-
tion of the crosslinking degree in the click-gels [247,248].
This novel procedure for the in situ rapid chemical gela-
tion of aqueous solutions of hyaluronan was employed.
Briey, water-soluble polysaccharide derivatives bearing
side chains endowed with either azide or alkyne terminal
functionality were prepared. When the latter two types of
derivatives are mixed together in aqueous solution they
give rise to a 1,3-dipolar cycloaddition reaction resulting
in fast gelation at room temperature. In the
1
H HR/MAS
spectra the signals of the HA moiety are clearly identied
together with the signal of the triazole ring formed during
the crosslinking reaction. It is possible to obtain a semi-
quantitative estimate of the crosslinking degree in the gels
by integrating the signal at 2.00ppmdue to the acetyl pro-
tons of HA, with respect to the resonance at 7.88ppmdue
to the triazole ring proton.
The structure and quantity of crosslinks in
chitosan/diethyl squarate (DES), chitosan/1,1,3,3-
tetramethoxypropane (TMP) and carboxymethylcellulose-
hydroxyethyl cellulose/divinylsulphone (DVS) networks
were investigated by
13
C CP/MAS NMR in the compre-
hensive work of Capitani, Crescenzi and co-workers
[239,240,242,243,252,253]. DES may be thought to act
both as a bidentate crosslinking agent and as a mon-
odentate substituent, reacting with compounds having
primary amino groups, including simple aminosugars.
With chitosan, the reaction takes place in a single step
under mild conditions.
The similar estimation of the crosslinking degree
with
13
C CP/MAS was performed for superabsorbent
carboxymethylcellulose-hydroxyethylcellulose/DVS
hydrogels by Lionetto et al. [249]. The number of crosslinks
per monomer was calculated fromthe ratio between one-
half of the area of signal at 56ppm, which is due to the
methylene carbon adjacent to the sulfoxide group, and the
sumof the area related to peaks associated with anomeric
carbons present in polysaccharide rings as a reference.
Table 2
Percentage of squarate bound to chitosan in the samples with a different
crosslinking degree R
s
as detected with
13
C CP-SPI/MAS [240].
R
s
% of squarate bound to chitosan
0.43 0.15
0.60 0.23
0.86 0.32
The resulting degree of crosslinking was 0.04mol per
mole of the polysaccharide ring, which corresponds to
1.7610
4
mol/cm
3
.
A partial spectral assignment of the
13
C CP/MAS spec-
tra was obtained through a spectral editing sequence of
SPI, which was proposed by Wu and Zilm[254]. The tech-
nique is the standard cross-polarization combined with a
simultaneous phase inversion (SPI) during the polariza-
tion inversion (PI). Properly choosing the pulse lengths
for the SPI, z
PI1
and z
PI2
, a
13
C spectrum can be obtained
where methine signals disappear and methylene peaks are
inverted, while signals due to methyls and non-protonated
carbon atoms are still positive.
The
13
C CP/MAS spectrumof a dried chitosan/DES gel, at
a stoichiometric crosslinking degree of R
s
=0.6, is shown in
Fig. 17a. In the range of 160200ppm, many resonances
were observed due to carbonyl and quaternary carbon
atoms. ByapplyingtheselectiveCP-SPI sequence, thesesig-
nals appear to increase their resolutionandcanbe assigned
(Fig. 17b) [240]. Here all signals of methine carbon atoms
are zeroed while, at 57ppm, with the phase fully reversed,
the methylene C
6
resonance is unequivocally assigned.
Table 2 shows that by increasing R
s
an increasing amount
of bound squarate was detected.
The similar investigation was performed for the
crosslinked hydrogels of carboxymethylcellulose (CMC),
which were analyzed with
13
C and
15
N CP/MAS [252,253].
Two types of hydrogels were prepared via Passerini and
Ugi condensation. In the Passerini three-component con-
densation, a carboxylic acid and a carbonyl are condensed
with an isocyanide, which after acyl rearrangement and
tautomerization, yields an-acyloxyamide. Inthe Ugi four-
component condensation the reaction mixture contains
an amine which condenses with the carbonyl to yield an
imine. The protonatedimine andthe carboxylate react with
the isocyanide to give -acylamineamide.
In the
13
C CP/MAS spectrum of the network with a
theoretical degree of crosslinking of 12%, obtained by the
Passerini condensation, signals frommethylene carbons of
the cyclohexyl units and aliphatic methylenes C
11
, C
12
and
C
13
at 2040ppmwere observed. The signal at 50ppmwas
ascribed to the methine carbon of the cyclohexyl units. The
methine carbon C
9
resonates in the range of 7080ppm,
i.e., in the same range as most of polysaccharide carbons
and cannot be detected. The broad signal at 172ppm was
due to the ester and amide carbons C
8
and C
10
respectively,
while at 179ppm the signal of the carboxylate was still
observed. As expected, increasing the theoretical degree of
crosslinking to 24%, an increase of the intensity of the car-
bon signals pertaining to the crosslink of approximately a
factor of 2was observed. Tofurther conrmthe assignment
in the
13
C CP/MAS spectrum, the SPI sequence was applied.
Inthis case, the methylene carbonsignals inthe 2040ppm
Fig. 17.
13
C CP/MAS spectrum of a dried chitosan/squarate network at
R
s
=0.6 before (a) and after (b) applying the CP-SPI sequence. All signals of
methane carbons are zeroed, while the inverted signal of the methylene
carbonC
6
is observable. Signals of methyl, carbonyl andquaternarycarbon
atoms are positive [239]. Copyright permission fromAmerican Chemical
Society, 1998.
range were inverted; signals due to the methylene C
6
and
methylene carbons of substituents in positions 2, 3, and 6
resonating at 7475ppm were negative. The signal of the
methine carbon atomat 50ppmwas zeroed, while signals
of carboxylated carbons and the amide and ester carbons
at 179 and 172ppm, respectively, were positive.
IntheNMR spectraof CMC networks obtainedbytheUgi
reactionwithdegree of crosslinking of 12 and 24%the most
striking difference is in the intensity of signal at 50ppm.
The intense signal at 50ppmis due to the methine carbon
of the cyclohexyl and to the methylene carbons C
9
and C
11
attached to the nitrogen of the N,N-disubstituted amide in

the crosslink, whichare not present inthe Passerini gel. The
CP-SPI sequence conrms this assignment as shown by the
inversion of this signal.
Since CP is not a linear technique, the quantitative reli-
ability of intensity of signals in CP/MAS spectra must be
interpreted with caution. In fact the efciency of the CP-
process depends on the number of abundant proton spins
close to dilute
13
Cspins andontheir distance fromcarbons.
To solve this problem at least partially, the best con-
tact time should be chosen [51]. For the above-mentioned
gels, the best contact time of 1ms was found [239]. The
semi-quantitative data can be obtained from the cross-
polarization dynamics as
S(t) =
S
0
z
_
1 exp
_
zt
1
IS
__
exp
_
t
1
1
(
1
H)
_
z = 1 +
1
IS
1
1
(
13
C)

1
IS
1
1
(
1
H)
,
(18)
where S
0
is the intensity of the investigated signal at the
contact time t =0, T
1
(
1
H) and T
1
(
13
C) are the proton and
the carbon spinlattice relaxation times in the rotating
frame and T
IS
is the cross-relaxation time between protons
and carbons. In homogeneous systems in which T
1
(
1
H)
is the same, the S
0
values are the true intensities of
the signals under investigation. When carbons have differ-
ent T
1
(
1
H) values, the MAS-SPE (single pulse excitation)
experiment is mandatory for a quantitative analysis [242].
In Fig. 18a the
13
C CP/MAS spectrum of a physical
mixture of CMC sodium salt and hydroxyethylcellulose
(HEC) powders, is shown [243]. At 104.0ppm the peak of
anomeric carbons C
1
of anyhydro d-glucose units unsub-
stituted in the C
2
position is observed, while at 97.8ppm
the signal of the anomeric carbon C
1(2*)
of units substi-
tuted in position 2 is observed. The shoulder at 82.7ppm
is mostly due to C
4
. The intense and broad signal centered
at 75.1ppm is due to C
2
, C
3
and C
5
, and the oxyethylene
chains of substituents. At 62.7ppmthe signal of unsubsti-
tuted C
6
carbon atoms is observed. In Fig. 18b and c the
spectra of the samples crosslinked with DVS at low (L)
and high (H) degree of crosslinking are shown. The peak
at 56.0ppm is due to methylene carbons of type A (see
Scheme in Fig. 18), i.e., to carbons adjacent to the sulfox-
ide group, while methylene carbons of type B resonate in
the same crowded range of frequency of C
2
, C
3
and C
5
, and
cannot be resolved. It is worth of note that the signal at
56.0ppmis absent in the spectrumof the non-crosslinked
sample (Fig. 18a).
In Fig. 19, the area of few selected carbon signals of
sample H are shown vs. the contact time. For each sig-
nal, S
0
and T
1
(
1
H) values have been obtained by tting
the experimental data to Eq. (18) (Table 3). The ratio R of
moles of reacted DVS and moles of saccharide rings can
be calculated from the ratio between a half of the area
Table 3
S
0
and T
1
(
1
H) values for the crosslinked CMC-Na/HEC hydrogel at high
degree of crosslinking (sample H) obtained by tting experimental data
to Eq. (18) [243].
Carbon S
0
T
1
(
1
H), ms
C
1
, C
1(2*)
12.01.0 6.00.3
C
4
12.01.0 5.50.4
C
6
6.60.5 5.00.3
A 6.50.5 4.60.3
Fig. 18. Schematic structure of CMC-Na-HEC crosslinked with DVS and
13
C CP/MAS NMR spectra of (a) a mixture of a non-crosslinked CMC-Na-
HEC powder (3/1), (b) sample L with lowdegree of crosslink, (c) sample H
with high degree of crosslink [243]. Copyright permission from Elsevier,
2003.
of the signal due to methylene carbons of type A, S
0
(A),
and the area of anomeric carbons C
1
and C
1(2*)
, S
0
(C
1
):
R=[S
0
(A)/2]/[S
0
(C
1
)]. The value of R for the case of sam-
ple H was shown to be 0.280.03. However, since T
1
(
1
H)
values of the peaks due to C
1
and Aare not strictly the same
(Table 3), MAS-SPE spectra were also performed. Chemical
shifts, line widths and areas obtained from these spectra
were used as an input in a programperforming a full spec-
tral deconvolution[243]. The evaluatedR(L) andR(H) equal
to 0.040.01 and 0.330.04mol of reacted DVS per mole
of saccharide ring, respectively.
The crosslinked HA-spermidine networks synthesized
by the Ugi reaction using formaldehyde, cyclohexyliso-
cyanide, and spermidine, were characterized by
13
C
CP/MAS NMR spectroscopy [252,253]. To conrmthe peak
assignment, the CP-SPI pulse sequence was applied. The
Fig. 19. Correlation between the areas of signals in the
13
C CP/MAS spec-
trumof sample H and the contact time t. Curves were obtained by tting
the experimental data to Eq. (18) [243]. Copyright permission fromElse-
vier, 2003.
area of the signal centered at 33ppm, due to the methylene
carbons 2 and 6 of the cyclohexyl rings and to the methy-
lene carbons and of spermidine, and the area of the
signal centered at 105ppm, due to the anomeric carbons of
HA, may be used for evaluating the real crosslinking degree
R. However, since
13
C CP/MAS spectra are not quantitative
the CP dynamics was carefully investigated for all the sam-
ples. Areas of the signals, S
0
, centered at 105 and 33ppm,
were reported as a function of the contact time. In this par-
ticular case, because the selected signals showed the same
T
1
(
1
H) values within experimental errors, the samples
were considered as homogeneous. As a consequence, the
obtained S
0
values can be used to quantitatively evaluate R.
The photo-switchable nitrocinnamate-modied PEG
hydrogel was synthesized via photo-crosslinking of the
nitrocinnamoyl groups [241].
1
H NMR spectra of photo-
crosslinked and photo-cleaved gel allowed estimation
of the crosslinking degrees. It is well known that only
trans-nitrocynnamide acid is capable of crosslinking by
formation of cyclobutane rings. Authors identied peaks,
which correspond to cis and trans isomers of nitrocin-
namic acid, as well as peaks of the cyclobutane ring, and
quantied the crosslinking. In the crosslinked gel, a dis-
tribution of 33% of trans- and 43% of cis-nitrocynnamic
acid and 24% of closed cyclobutane rings was detected.
This indicated that in parallel with the process of photo-
crosslinking the on-going trans-cis isomerization process
induced with UV irradiation occurred. A decrease in the
abundance of cyclobutane rings, which accounted for
19.8%, and the remaining trans-nitrocynnamic acid and cis-
isomer accounted for 36.4% and 43.9%, respectively, was
detected as a consequence of the photo-cleavage process.
In order to establish the structure of poly(amidoamine)
(PAA)-based crosslinked networks in the swollen state,
homo- and heteronuclear 1D (
1
H,
13
C, Jmod) and
2D HR/MAS (
1
H
13
C COSY, HMQC, HMBC) NMR spec-
troscopy was applied. The model crosslinked PAA sam-
ples with similar structures, but different crosslinking
degrees, obtained by polyaddition of 2-methylpiperazine
to 1,4-bis(acryloyl)piperazine with ethylenediamine as a
crosslinking agent were employed [246]. A complete
1
H
and
13
C assignment was performed with
1
H
13
C HMQC.
The
1
H spectra of the crosslinked samples revealed the
presence of a limited amount of vinyl functions cor-
responding to dangling chain ends. The vinyl protons
exhibitedtheir characteristic signals at 5.9, 6.4and7.1ppm.
The corresponding
13
C signals were found at 128 and
129ppm, and the assignments were conrmed by the CH
couplingconstant displayedinthe
1
H
13
CHMBCspectrum.
The higher mobility of the terminal groups (long T
2
) with
respect to the backbone units (short T
2
) allowed their easy
recognition, even though their concentration was limited.
The amount of vinyl dangling terminals was determined
by calculating the ratio between the integrals of vinyl car-
bon signals vs. the C
4
and/or C
5
of the polymer backbone.
The resulting percentage was about 4% of the overall num-
ber of 1,4-bis(acryloyl)piperazine-deriving units. The small
amount of vinyl dangling ends indicates that almost all the
NH functions participate in the polymerization reaction.
The intermolecular crosslinks were detected by com-
paring the spectra of linear and crosslinked PAAs. In
particular, the presence of methylene carbons of the
ethylenediamine moieties was revealed by the correlation
of
13
C signal at 50.5ppm in the
1
H
13
C HMQC spectrum,
with the protons at 3.55ppm. This cross-peak was absent
in the
13
C spectrum of the linear PAA and its intensity
increased along with increase of degree of crosslinking.
The crosslinking degree could not be directly determined
by
13
C NMR from the integral of the peaks relative to the
CH
2
of the ethylenediamine moieties because they were
broad. An alternative procedure was therefore adopted
[246]. The values, determined from the integral ratio of
the CH
3
(16.8ppm) of 2-methylpiperazine and the C
3
and C
6
carbonyl signals (170.4 and 170.7ppm) of 1,4-
bis(acryloyl)piperazine were 0.81 and 0.59, respectively,
that is very close to the feeding. Therefore, ethylenedi-
amine participates in binding the stoichiometric excess
1,4-bis(acryloyl)piperazine over 2-methylpiperazine.
2.6.3. Mechanisms of crosslinking by gelation
The chemical crosslinking of gel networks can be ful-
lled either with the aid of irradiation or a free-radical
initiator; without or with the aid of a crosslinking agent.
In order to establish mechanism of crosslinking,
13
C
CP/MASNMR spectra of freeze-driedcyclohexanegels from
diacetylene cholesteryl esters with two urethane linkages
were obtained before and after UV irradiation [255]. After
irradiation, the intensity of the peaks at 65.6 and 77.9ppm
from interior and exterior sp-hybridized carbon atoms
of the monomeric diacetylene unit decreased and new
peaks assigned to the sp- and sp
2
-hybridized carbon atoms
of the polydiacetylene backbone appeared at 106.3 and
130.5ppm. No other new peaks for sp- or sp
2
-hybridized
carbons are observed after irradiation. This implies that the
1,4-addition polymerization reaction prevails in the course
of gelation.
Mechanism of the UV-initiated oxidative crosslinking
of poly(N-vinylpyrrolidone) (PVP) hydrogels was studied
by FTIR, Raman and
13
C CP/MAS techniques [256]. Both
FTIR and Raman spectra indicated that in the course of
crosslinking, the pyrrolidone ring is partially transformed
into a succinimide ring. Solid-state NMR data have con-
rmed this change, and showed that stable intermediates
of 4-hydroperoxy- (81.6ppm) and 4-hydroxy-pyrrolidone
(87.5ppm) are formed. The hydroperoxide is responsive for
efcient crosslinking. This investigation is a good example
of the benecial combining of the various spectral methods
to sort out with the mechanismof chemical crosslinking.
13
C CP/MAS technique was also applied for charac-
terization of crosslinks and bond-scission produced by
-irradiation in poly(-glutamic acid) (-PGA) hydrogel
[257]. After -irradiation, thepeaks frommethine(73ppm)
and methylene (68ppm) carbons bonded to oxygen or
nitrogen appeared; most possible crosslinks and hydrol-
ysis by radical reactions are supported by formation of
chemical bonds of CH
2
N, CH
2
O, CHN, and CHO. It was
shown that in the course of irradiation, not only crosslink-
ing but alsobond-scissioncanhappen. The carbonyl carbon
is involved in the crosslinks and hydrolysis can be taken
into account to explain the small peak at 174.4ppm.
The crosslinking in poly-(N-isopropylacrylamide)-clay
nanocomposite (PNIPAAm-clay NC) hydrogels was inves-
tigated by light scattering, uorescence correlation spec-
troscopy and
1
H NMR [258]. The UV-photopolymerization
was chosen for the free-radical crosslinking. Experiments
showed that shortly before the gelation threshold is
reached, no changes in the light scattering autocorrelation
functions appear, while the monomer conversion can be
observed by
1
HNMR spectroscopy. The kinetics of gelation
process of the NC gels was further monitored by
1
H NMR.
1
H HR/MAS NMR spectroscopy was instrumental to
monitor a conversion of vinyl and some other polymeriz-
able groups in the course of free-radical polymerization
of poly(2-acrylamido-2-methylpropanesulphonic acid-
co-N-isopropylacrylamide) chains crosslinked with
methylene bisacrylamide, with interpenetrating linear
chains of PEG [259], dimethylacrylamide (DMAAm)
with PEG diacrylate as the crosslinker in a semi-frozen
aqueous solution [260,261], methacrylated inulin [230],
poly(acrylamide-co-acrylic acid) [262], or PEG-diacrylate
macromer crosslinked with ammonium persulfate or
N,N,N
-tetramethylenediamine as initiators [263],

branched PEG-succinimidyl propionates crosslinked with
different types of PEG-amines [264], PEG-octavinylsulfone
crosslinked with PEG-octaamine [265], oligo(ethylene
oxide) monomethyl ether methacrylate (OEO
300
MA) with
poly(ethylene oxide) dimethacrylate as a crosslinker
[266] and methacrylated chitooligosaccharide chains
[267], dextrin, grafted with vinyl acrylate [268], N-
acryloyl-tris-(hydroxymethyl)aminimethane crosslinked
with poly(2-methyl-2-oxazoline) bismacromonomer,
methacrylic adduct of gelatin [237], ethylene glycol
dimethacrylate or N,N
-methylenebisacrylamide [269]
followed by formation of the respective hydrogels. The
signals from vinyl groups at 5.76.9ppm were the most
suitable for monitoring the conversion based on decrease
in their intensity. The similar investigation with aid of
13
C CP/MAS based on disappearance of signals from C C
groups at 120140ppm and appearance of a new signal
from non-conjugated C O groups at 170188ppm was
performed for hydrogels from arabic gum, pectin and
chondroitin sulfate chemically modied with glycidyl
methacrylate [231235]. It was shown in all these cases
that crosslinking of polymer chains takes place predomi-
nantly at the early stages of chain growth, and the crosslink
density of the network decreases with increasing agent
conversion.
These kinetic studies allow making the important con-
clusions about the mechanism of gelation. For example,
the presence of a very broad distribution of polymer
sizes during crosslinking is expected, but the distri-
bution is likely different from that predicted by the
FloryStockmayer theory [28]. This was particularly evi-
dent fromthe high end-group conversion measured by
1
H
NMR, which indicates signicant intramolecular crosslink-
ing (i.e. ramication). According to the FloryStockmayer
theory, monomers and dimers should predominate at the
gel point. If all the end-groups were equally reactive, any
single particle that grewto a signicant size would rapidly
subsume the other molecules. Besides, molecular dynam-
ics simulations showed that chain exibility alone leads
to deviations from the FloryStockmayer theory, which
result in microgel formation [270]. Monte Carlo simula-
tions of hydrogel formation were performed to assess the
size distributions that result in decreasing reactivity with
increasing chain size. Indeed, NMR data indicated that
the rate of reaction increased exponentially as reactive
groups were consumed. Clearly, intramolecular crosslink-
ing is signicant, as conversionof 0.64 was observed before
gelation. This is possible only if intramolecular reactions
dominate intermolecular reactions, since the gel point
would otherwise be expected at conversion of 0.14.
The mechanism of synthesis of superporous hydrogel,
SPH, and SPH composites was studied with
13
C CP/MAS
NMR [262]. The spectra showed that the SPH polymer
was made of acrylamide (AAm) and acrylic acid (AAc)
monomers as a backbone chain, which was crosslinked
with N,N
-methylenebisacrylamide. Radicals, formed after

free-radical initiation, attack the double bonds of AAm
and AAc, and also to a less extent the double bond of
N,N
-methylenebisacrylamide following by formation of

the long aliphatic chains. These chains are subsequently
crosslinked by the added crosslinker. Concentration of the
crosslinker controls the densityof crosslinks betweenpoly-
mer backbone chains.
13
CCP/MASNMR was appliedtocharacterizethemolec-
ular structure of the hydrogel fromalginate modied with
glutaraldehyde [236]. The spectrum of the hydrogel was
similar to that of the native alginate, except for the inten-
sities of peaks at 68 and 75ppm(corresponding to G2 and
M3, M2 and G3; G guluronic and M mannuronic acids),
whichdecrease relative tothe G5signal. The decrease is the
result of crosslinking of the two hydroxyls at carbons 2 and
3 in mannuronic and guluronic acids with glutaraldehyde,
forming acetal linkage, R
2
C(OR
)
2
, which has a charac-
teristic peak at 105ppm. G2, G3, M2, and M3 are being
converted fromhydroxyl-bearing carbons to ether-bearing
carbons, which results in a chemical shift change and a
broadening of the collective signals for these carbons rela-
tive to G5, G6, M5, and M6.
A self-assembling l-glutamide-derived lipid with a
triethoxysilyl headgroup (Si-lipid) was synthesized to sta-
bilize the chirally ordered state of the nanobers in
various organic solvents [271]. Polycondensation of the
triethoxysilyl groups was carried out by acid-catalyzed
hydrolysis and condensation in a benzene gel and con-
rmed by
29
Si CP/MAS NMR and FTIR measurements. In
the
29
Si CP/MAS NMR spectra of freeze-dried benzene gels,
only one strong signal at 46.4ppmof triethoxysilyl group,
RSi(OEt)
3
, was detected before the condensation reac-
tion. It disappeared after the condensation, and three new
signals appeared at 49, 58, and 67ppm, which were
assigned to monocondensed (RSi(OSi)(OH)
2
, P1), dicon-
densed(RSi(OSi)
2
(OH), P2), andtricondensed(RSi(OSi)
3
,
P3) structures, respectively. Because the
29
Si signal of the
hydrolyzed trihydroxysilyl group, RSi(OH)
3
, appears at
38ppm, it is conceivable that the condensation reactions
mostly occurred among Si-lipid aggregates. The P1 and
P3 structures act as a terminal group and a crosslinking
point, respectively. The peak ratio of the three silicone
species related to the degree of polymerization and chem-
ical crosslinking of poly(Si-lipid) with 0.125 equiv of TFA
(P1:P2:P3=15.9:64.0:20.1) was close to that obtained with
0.25 equiv of TFA (P1:P2:P3=16.6:61.3:22.1). The P1 com-
ponent increased and the P3 component decreased in
the poly(Si-lipid)/benzene gel obtained with 0.025 equiv
of TFA (P1:P2:P3=25.5:60.2:14.3). This indicates that the
degree of polymerization and the number of crosslinking
points decreased in the poly(Si-lipid) obtained with less
TFA. These results are consistent with the gelation prop-
erties of Si-lipid after polycondensation. It is clear that the
diameters of brous aggregates increased after polycon-
densation.
Crescenzi et al. [247,248] obtained hydrophilic net-
works based on functionalized hyaluronic acid and on a
partially acetylated chitosan. Crosslinking reactions were
performed using glutaraldehyde. The obtained networks
were qualitatively characterized by means of
13
C CP/MAS
technique. The broad signals observed in the 2648ppm
range were due to different types of methylene carbons of
glutaraldehyde which belong to the crosslinks. Weak and
broad signals in the 120150ppm range were due to car-
bon atoms of C C double bonds and quaternary carbons of
glutaraldehyde.
In the more comprehensive kinetic experiment, the
1D
1
H DOSY spectroscopy was used to characterize
vinyl conversion of poly(ethylene glycol) (PEG) and
glycolide macromonomers into poly(ethylene glycol)-co-
poly(glycolic acid) (PEG-co-PGA) hydrogels after pho-
topolymerization [272]. Oligo(glycolic acid) was poly-
merized from both ends of the bifunctional PEG block
to provide hydrolysable linkages.
1
H DOSY spectroscopy
allows improving the resolution of peaks and charac-
terizing the efciency of vinyl group reactivity during
photo-crosslinking. The conversion was evaluated by
comparing the relative peaks of non-crosslinked and
crosslinked methylene protons.
The mechanism of crosslinking between biopolymers
containing primary amine groups (chitosan, BSA and
gelatin) with genipin was investigated by FTIR, UVvis, and
13
C NMR spectroscopies, protein-transfer reaction mass
spectrometry, and dynamic oscillatory rheometry [273].
It was shown that two separate reactions led to forma-
tion of crosslinks between primary amine groups. The
13
C NMR kinetic data, based on decrease in intensities
of genipin peaks, showed that the fastest, and, there-
fore, rst reaction to occur was a nucleophilic attack of
the genipin C
3
carbon atom from a primary amine group
that led to the formation of a heterocyclic compound of
genipin linked to the glucosamine residue in chitosan and
the basic residues in BSA and gelatin. The slowest sec-
ond reaction was the nucleophilic substitution of the ester
group possessed by genipin to release methanol and form
a secondary amide link with chitosan, BSA, or gelatin.
Measurements detectedthe second-order gelationkinetics
resulting froman irreversible crosslinking process. Protein
secondary and tertiary structures were important in deter-
mining the availability of sites for crosslinking in protein
systems because a lower elastic modulus G
was attained
after a given time during crosslinking of the globular pro-
tein BSA than the coiled protein gelatin, despite possessing
more crosslinkable basic residues.
The crosslinking of gelatin was thoroughly studied by
13
C NMR in both the solution and solid state using
13
C
enriched caffeic acid (LCA) as the crosslinking agent [274].
To determine whether the formation of CN linkages
between LCA and gelatin occurs, the gelatin/LCA systems
were examined by
13
C CP/MAS. With a CP contact time of
1ms most signals were detected for the gelatin/LCA gels.
However, only protonatedcarbons canbe observedat short
contact times of 20s. This conrmed that resonances at
130 and 145ppmhad no bonded hydrogen, the same as the
C O in gelatin and phenolic COH carbons of LCA, corre-
spondingtotheimplicit formationof CNlinkages between
LCA and gelatin. In contrast, using 10ms spin-locking time
in
13
C channel before acquisition detects only carbons
with longer
13
C T
1
, that are, normally, non-protonated
carbons.
The
1
HT
1
data of different protons ingelatinwereiden-
tical, indicating strong spin diffusion within the gelatin
matrix which is homogeneous at the scale of the spin-
diffusion path length of 23nm within the T
1
time. This
suggests that LCA is homogeneously distributed within
the gelatin matrix at the scale of 23nm and the whole
gelatin/LCA gel is homogeneous with the domain sizes
(such as crosslinked regions, plasticized phases, etc.)
smaller than this scale.
A principally new approach for determination of the
mesh size and crosslink density
c
based on the MQ
NMR spectroscopy was proposed by Saalwchter and co-
workers [116,275279]. It is known that the presence of
crosslink-inducedresidual dipolar couplings (D
res
) ina net-
work leads to the accelerated relaxation as compared to
a normal liquid. The magnitude of D
res
is inversely pro-
portional to the chain length between the crosslinks and
thus directly proportional to
c
[116]. The intensity decay
in a relaxation experiment is in this case due to coherent
spin evolution (dipolar dephasing) rather than fast-motion
induced relaxation. Since true relaxation processes always
act simultaneously with the dipolar dephasing, making
analyses of relaxationcurves model-dependent, it is advan-
tageous to measure the residual dipolar couplings directly
in an experiment where their presence leads to a quanti-
able intensity build-up rather than decay. In this respect,
MQ NMR has evolved as one of the most quantitative
approaches, because not only absolute values of D
res
, but
also their distribution can be measured [116].
Normally, the MQ NMR experiment yields double-
quantum (DQ) intensity, I
DQ
(z
DQ
), that depends on the
product of D
res
and the evolution time, z
DQ
. In a refer-
ence experiment, intensity, I
ref
(z
DQ
), is measured, which
belongs to that part of the total magnetization that has
not yet evolved into DQ coherences. It contains a com-
plementary contribution fromdipolar coupling modulated
magnetization of the network, and also all contributions
from more mobile non-network components. The sum
of DQ and reference intensities, I
lMQ
=I
DQ
(z
DQ
) +I
ref
(z
DQ
),
was normalized. Then, a contribution termed C-fraction
couldbeseparatedbyttingthelong-timetail of thedecay-
ing function to a single-exponential function with initial
amplitude f
C
and relaxation time z
C
. The C-fraction cor-
responds to very mobile components such as solvent and
sol that have the longest relaxation time. It is not of major
interest and was subtracted fromI
lMQ
. A second contribu-
tion (B) that is polymeric in nature but elastically inactive
and can be associated with dangling chains, loops and
related structures, has to be subtracted as well [275279].
The reliable tting of this contribution, which also decays
exponentially, is challenged by the fact that the corre-
sponding relaxation time z
B
is very close to the relaxation
time of the I
lMQ
(z
DQ
) intensity of the pure network com-
ponent (A) under the given conditions, even though this
component is not network-like and does not contribute to
the DQintensity build-up. This is so because the ultimately
isotropic segmental motions of this component occur on a
timescale that is similar to that of the network chains, as
opposed to the faster and isotropically mobile sol part C.
In this case, an alternative approach had to be taken that
makes use of the fact that the nal DQ intensity of the
network part has to evolve to the same level as the cor-
rected reference intensity, i.e., I
DQ
/I
lMQ,corr
I
nDQ
=0.5 as
z 0.5 [116]. This condition was met by multiplying the
C-correctedsumintensityI
lMQ
(z
DQ
) byafactor, 1f
B
, cho-
sen such that the so-called normalized DQ build-up curve,
I
nDQ
(z
DQ
), approaches the theoretically expected value of
0.5 in its long time limit. The contribution of effective net-
work chains (A) to the total signal intensity is then simply
given by f
A
=1f
B
f
C
.
A listing of the fractions of the three components A, B,
and C, as well as their apparent relaxation times at 25
C
for PAAmfunctionalizedwithdimethylmaleimide (DMMI),
are shown in Table 4. z
C
values are in the range of several
hundred ms, while the common z
A
=z
B
is 10ms. Besides
a roughly constant portion of about 20% sol and solvent
Table 4
Results of MQ NMR experiments on semi-dilute samples of PAAm-DMMI
in the crosslinked state [278].
Concentration (g/L) f
A
(%)
20 11.5 56.5 32.0 14.2 1369.9 85.1
40 32.4 48.6 19.0 11.1 404.9 109.6
60 40.4 40.4 19.2 10.4 423.7 128.9
80 44.0 31.8 24.2 8.9 251.9 127.2
f
A
, f
B
and f
C
are the fractions of network chains, dangling chains, etc., and
sol, respectively, z
A
, z
B
and z
C
are corresponding relaxation times, D
res
is
the residual dipolar coupling. All data obtained in D
2
O at 25
C.
Fig. 20. Normalized DQ build-up curves obtained fromMQ NMR spectra
of semi-dilute PAAm-DMMI samples in their fully crosslinked states at
various concentrations of 20triangles down, 40triangles up, and80g/L
squares [278]. Copyright permission fromElsevier, 2007.
in all samples, f
A
is distinctly increasing with rising poly-
mer concentration on the expense of f
B
. This is another
indication of the fact that the higher the polymer concen-
tration, the more is the formation of elastically effective
crosslinks favored over the formation of intramolecular
linkages leading to loops, etc. f
A
is thus considered a mea-
sure of crosslinking efciency.
Fig. 20 demonstrates the DQ build-up curves of the
network component in the gel samples, obtained after nor-
malization. The build-up of I
nDQ
with z
DQ
depends on D
res
in the following way:
l
nDQ
(D
res
) =
1
2
_
1 exp
_
_
2
5
_
D
2
res
z
2
DQ
__
(19)
D
res
in turn is, as mentioned above, inversely related
to the apparent chain length between restrictions [275].
In general, a distribution of chain lengths and, hence, a
distribution of coupling constants needs to be consid-
ered. For the evaluation of data, a gamma distribution
was assumed as discussed in detail [116]. It is ideally
expected in a system with xed chain length between
crosslinks and a Gaussian distribution of end-to-end
distances:
P(|D
res
|) =
2
_
27|D
res
|
8D
3
res
exp
_
3|D
res
|
2 -D
res
:
_
(20)
The experimental I
nDQ
(z
DQ
) data were then tted in the
region of I
nDQ
<0.45 by integrating over the entire range of
D
res
=0 . . . to give a mean value of a relatively broad dis-
tribution. Results of these ts are shown in Fig. 20 as solid
lines, and the average coupling constants are included in
Table 4 [278]. The coupling constants and the courses of
I
nDQ
(z
DQ
) are quite similar for all PAAm-DMMI gels, despite
of different concentrations and crosslinking efciencies.
This indicates that the mean lengths of network chains are
similar, and a consideration of the crosslinking mechanism
can explain this nding: a linkage between two polymer
chains is only formed upon dimerization of two DMMI side
groups and, therefore, the chain length between crosslinks
(and its distribution) is essentially predetermined by the
given random spacing of DMMI moieties along the initial
polymer chains [278,279].
The experimental data also imply that dimerization
which does not generate elastically active network chains
does not lead to chain extension to any appreciable extent.
This is asurprisingandimportant observationinviewof the
marked variation of the A fraction determined by MQ NMR
or the crosslinking efciency, respectively, with polymer
concentration. The necessity to assume a broad distribu-
tion of coupling constants according to Eq. (20) in order to
attain reasonable tting of the build-up curves is in accord
with the previous observations on swollen networks [276].
While distributions due to variations in the chain length
between crosslinks as well as the distribution expected on
the basis of a Gaussian distribution of end-to-end distances
are screened by the cooperativity of the chain motions
(packing) in unswollen hydrogels, such effects, as well as
topologicallyinducedswellingheterogeneities, reappear in
swollen networks [116].
2.6.4. Swelling and deswelling
In order to reveal the swelling mechanism, the non-
ionic polymethyloxazoline hydrogels were synthesized by
the cationic ring-opening copolymerizationof 2-methyl-2-
oxazoline and 2,2
-tetramethylenebis(2-oxazoline), using
random copolymers of chloromethylstyrene and MMA, or
of chloromethylstyrene and styrene as macroinitiators, by
radical homopolymerization of vinyl end-functionalized
poly(2-methyl-2-oxazoline) bis(macromers), or by rad-
ical copolymerization of these bis(macromers) with
N-vinyl-2-pyrrolidone [280]. The hydrogel structures were
characterized by
1
H HR/MAS NMR, and their solvent
absorption capacity was determined by swelling experi-
ments in solvents of different polarity. The study allowed
assuming the formation of rigid domains by the hydropho-
bic macroinitiator backbone, which can provide increased
mechanical stability and which corresponds well also with
the observed solvent swelling behavior.
Philippova et al. [281] followed by the
13
C NMR split-
ting of signals of n-alkyl groups during ionization for the
hydrophobically modied pH-responsive PAAc hydrogels,
containing up to 20% mole of n-alkyl acrylate units ran-
domly distributed along the network chain, in order to
measure the fractionof hydrophobic groups includedinthe
hydrophobic clusters. To fulll the comparable conditions
with the equilibriumswelling curves and the uorescence
data, the gel samples were prepared, at given values of
ionization degree . At 0.2, only one peak for each
carbon group ([CH
2
] =23.5ppmand [CH
3
] =14.9ppm) is
present. These peaks were attributed to the aggregated
formof n-alkyl chains. At a further increase of to 1.0, new
peaks corresponding to the non-aggregated CH
2
and CH
3
groups appear at 23.1 and 14.5ppm, respectively. Increase
in ionization degree leads to an increase of the relative
intensity of these peaks. Thus, the gel ionization results in
a partial disruption of hydrophobic aggregates. But even
at =1, it was estimated that ca. 40% of the n-alkyl chains
of the hydrogel remain embedded inside the hydrophobic
clusters.
The effects of continuous water exchange on the
swelling behavior of poly(N-isopropylacrylamide-co-
sodium acrylate), poly(NIPAAm-co-SA), gel were studied
[282]. To this effect,
13
C CP/MAS, the solid-state
1
H com-
bined rotation and multiple-pulse spectroscopy (CRAMPS)
and swollen-state
13
C dipolar decoupled/MAS (DD/MAS)
experiments were carried out for dried and swollen sam-
ples by varying the time of water exchange. As a result, the
intensity and position of the carboxyl peak changed, and
the relative intensity of the non-ionized carboxyl groups
of gels increased with an increasing number of water
exchanges. These results indicated that hydrogen bond-
ing was formed between the two non-ionized carboxyl
groups of SA and/or between the carboxyl and carbamide
groups. The macroscopic polymer network shrinkage was
discussed in terms of the replacement of counterions Na
+
by H
+
followed by intermolecular H-bonding.
The variable-temperature time-lapse
1
H NMR spec-
troscopy was performed to obtain the molecular level
insights into dehydration kinetics of the hydrogels [283].
The spectra were then tted to a sum of four Lorentzians,
and the decrease in peak heights was used as a mea-
sure of the peak broadening. At a given temperature, the
magnitude of dehydration retention time was inversely
proportional to the swelling ratio, which was, in turn,
proportional to the degree of crosslinking. This was
because H-bonding in a more densely crosslinked hydro-
gel is more resistant to disruption. The dehydration of
polymer chains was always much faster than the macro-
scopic deswelling. The dehydration rate at molecular level
increased with temperature, in contrast to the rate of
macroscopic deswelling.
Summarizing, a puzzling contradiction may appear
upon analysis of the NMR data. The chemical shift variation
clearly indicates that discrete molecules are observed, and
the reduced tumbling rates detected by relaxation mea-
surements and NOE experiments suggest the presence of
oligomeric assemblies. The possibility that an increase in
viscosity would result in the variation of the T
2
values can
be excluded since some species do not change their relax-
ation times upon gel formation. It has also been shown
that the line width of solvent signals in supramolecular
gels is not affected by gel formation, revealing that the
macroscopic viscosity increase does not affect the tum-
bling rates of the solvent in accordance with the presence
of large pools of solvent within the gel [34,47,48,132]. The
invariance of chemical shifts and modication of tumbling
rates (T
2
values) upon gel formation is not unique and this
issue has been addressed by Duncan and Whitten [34].
They pointed out the above-mentioned contradictory facts
and concluded that in their systemfast exchange between
mobile gel regions and free organogelators exists. The
disturbing observation that no chemical shift variation is
observed upon gel formation can be explained on the basis
of the fact that for mobile gel regions accessible to solvent
smaller shifts would be anticipated.
3. NMR spectroscopy on the molecular dynamics of
hydrogels and organogels
Among the various experimental techniques used to
this aim, NMR has proven to be unique in characteriz-
ing the dynamics of gelator aggregates and incorporated
solvent molecules, particularly through measurement of
1
H and
13
C spinlattice (T
1
) and spinspin (T
2
) relaxation
times as well as self-diffusioncoefcients D[6,15,39]. How-
ever, it must be borne in mind that in complex systems
such as hydro-/organogels analysis of relaxation data is
not straightforward since magnetization transfer between
polymer and solvent nuclei via chemical exchange and/or
cross-relaxation must be taken into account [170173].
The NMR spectroscopy can also give site-specic informa-
tion on the inuence of hydration on polymer dynamics
through
13
C cross-polarization (CP) and direct excitation
(DE) MAS experiments. Whereas the former enhances
signals arising from carbons strongly dipolar-coupled to
protons, typically
13
C atoms directly bonded to protons
and in a rigid environment, the latter, performed with a
very short recycle delay (on the order of seconds), shows
only fast relaxing carbonnuclei, usually locatedinthe quite
mobile environment.
For example, recently the temperature-dependent
13
C
NMR spectra of hydrogels from poly(acrylate)s, poly(2-
methoxyethyl acrylate) (PMEA), poly(2-hydroxyethyl
methacrylate) (PHEMA), and poly(tetrahydrofurfuryl acry-
late) (PTHFA), were obtained with DD/MAS and CP/MAS,
to gain insight into their network structures and dynamics
[284]. The DD/MAS yields high-resolved
13
C NMR spec-
tra due to the presence of high frequency motions of
swollen exible, polymer chains (>10
5
kHz) [4,5], although
the corresponding CP/MAS spectra were consequently
suppressed. In contrast,
13
C NMR signals from inexible
chains including crosslinked region could be recorded
by CP/MAS. In this case, it is emphasized that such low-
frequency motion in the polymer chains could be detected
by suppressed peak intensities. For this purpose, authors
examined suppressed or recovered intensities (SRI) plots
of DD/MAS and CP/MAS spectra, caused by interference or
escape of incoherent uctuation frequency with coherent
frequency of proton decoupling or MAS, respectively, to
reveal occurrence of such low-frequency motions.
Fig. 21a andbshowthe
13
CCP/MASandDD/MASspectra
of PHEMA hydrogel at various temperatures. The maxi-
mumpeak intensities were available fromthe CP/MAS and
DD/MAS spectra recorded at the lowest and highest tem-
peratures, respectively, consistent with the expected SRI
plots [284]. Fast isotropic motions withfrequency>10
5
kHz
were located to the populations in which
13
C CP/MAS
signals of swollen PMEA were selectively suppressed. In
contrast, low-frequency motioninthe range of 10100kHz
was identied to the populations in which
13
C DD/MAS
(and CP/MAS) signals are most suppressed at the char-
acteristic suppression temperature T
s
. It was found that
exibility of gel network (PMEA>PTHFA>PHEMA) is char-
acterized by T
s
(PMEA<PTHFA<PHEMA).
3.1. NMR relaxometry and gel dynamics
In hydro-/organogels, both the nonselective and selec-
tive NMR relaxation times of various proton types can be
measured. To deduce fromthe NMR behavior any informa-
tion on the phase composition, structure and dynamics, it
is instructive to derive a general theoretical expressionthat
relates the relaxation rate temperature dependencies of a
Fig. 21.
13
C CP/MAS (a) and DD/MAS (b) NMR spectra of PHEMA hydrogel containing 40wt.% of water recorded at various temperatures. The assignment
of PHEMA carbons is done according to the formulas at the top [284]. Copyright permission fromElsevier, 2009.
solute to changes in its environment. The dominant NMR
relaxationpathwayfor solute protons at loweldstrengths
is mediated by proton dipolar interactions. These include
both intra- and intermolecular contributions [40].
3.1.1. General consideration
The expression for the relaxation rate of a single aggre-
gate phase can be written as generalization of Eqs. (4) and
(6) as follows [34]:
R
i
= K
2
_
_
j / = i
r
6
ij
j
ij
(, 1) +
ij
(r
ij
, , 1)
_
_
, (21)
where K is a collection of constants for the dipolar
interaction, is the magnetogyric ratio and the two
terms reect intramolecular and intermolecular contribu-
tions, respectively. The only difference in the expressions
between R
1
and R
2
rate constants is in the shape of
the spectral density. The spectral density j
ij
(, 1) arises
from magnetic eld uctuations generated by molecu-
lar motions within the vicinity of the dipolar-coupled
nuclei and serves to modulate the dipolar interaction. The
intermolecular term j
ij
(r
ij
, , 1) is summed over spins j
residing on neighboring solute molecules that are dipolar-

coupled to the observed spin i. The dipolar distance, r
ij
,
is time-dependent because of translational diffusion [40].
The intramolecular spectral density terms of the non-
aggregated and aggregated gelator are different because
of the restricted motions. Furthermore, the spectral den-
sities of the intramolecular and intermolecular terms for
the aggregatedstate, j
ij
(, 1) andj
ij
(r
ij
, , 1), are different
because of signicant translational motion contributions
present in j
ij
(r
ij
, , 1) that are absent in j
ij
(, 1). In gen-
eral, the line-narrowing regime is obtained when is
much greater than the Larmor precession frequency,
0
.
Here the spectral densities in both R
1
and R
2
rates are
identical and narrow resonances are obtained as typical
for non-viscous liquids. However, as approaches and
decreases below
0
, as it is observed in solids and vis-
cous liquids, the weighting of the spectral density in R
2
to the low-frequency components becomes manifest by a
divergence in R
1
and R
2
rate behavior. The R
1
rate is max-
imal when
0
with an overall inverted parabolic-type
shape in plots of R
1
vs. J(). However, when <
0
, the
R
2
rate constants increase resulting in severely broadened
lines that become undetectable under conventional high-
resolution conditions [34].
From the thermodynamic point of view, both the
non-aggregated and aggregated gelator must coexist over
some temperature range. The aggregates include colloidal
structures within a liquid phase (sol), which should be
detectable in the high-resolution NMR spectra, and both
rigid (NMR silent) and mobile (NMR-detectable) struc-
tures within a brous solid network (gel). If only the
non-aggregated gelator is detectable with NMR, and no
exchange with aggregate structures occurs on the NMR
time scale, then the temperature dependence in R
1
and
R
2
can be described by the rst term of Eq. (21). Hence,
the similar behavior would be anticipated in a solvent
in which aggregation of gelator does not occur. Clearly,
if only a non-aggregated gelator is detectable, then the
observed R
1
and R
2
must reect the existence of an aggre-
gated gelator by an exchange process that is fast on the
NMR time scale. In this case the relaxation rates have a
single-exponential decay behavior. On the other hand, if a
gelator is NMR-detectable in both the non-aggregated and
aggregated states, then a fast exchange of gelator between
these states occurs because, in accord with Eq. (21), a
slowexchange would produce biexponential decay behav-
ior. A general expression for this behavior is illustrated in
Eq. (22) where the contributions fromthe non-aggregated
and aggregated states are weighted by their respective
temperature-dependent mole fractions of solvent, c
S
(T),
and, for generality, q different aggregated moieties with
mole fractions of c
p
(T), can be considered [34]:
R
i
= K
2
_
_
_
_
_
c
S
(1)
j / = i
r
6
ij
j
ij
(, 1)
_
_
solution
+
q
p=1
c
p
(1)
_
_
j / = i
r
6
ij
j
ij
(, 1)
_
_
+
_
_
ij
(r
ij
, , 1)
aggregate p
_
_
_
_
_
(22)
and c
S
(1) +
q
p=1
c
p
(1) = 1.
3.1.2.
1
H T
2
relaxation affected by an exchange
In heterogeneous gel systems, the observed
1
H NMR
spinspin relaxation is determined both by the intrinsic
relaxation of each of the proton populations and by effects
of magnetization transfer that may occur between them.
The variation of T
2
with temperature for hydrogels in the
presence and absence of chemical exchange of protons
between water and polymer is described [172,173] as:
1
1
2
=
1 p
c
1
2b
+
p
c
1
2c
+z
ex
, (23)
where T
2
is the observed value for the polymer/water
system, and p
c
is the ratio of the number of exchange-
able protons to the total proton pool, comprising water
protons, labeled the b species, and exchangeable poly-
mer protons, labeled the c species. T
2b
and T
2c
are the
intrinsic spinspin relaxation times of these two proton
populations, and z
ex
is the exchange correlation time. The
parameter p
c
is a constant that can be determined on the
basis of the composition and equilibriumwater content in
the hydrogel.
3.1.3. Anisotropic molecular motion
Bound or motionally restricted molecules may exhibit
motion on more than one distinguishable time scale. In
general, a distribution of correlation times is likely to be
more realistic representation of anisotropic motion. How-
ever, use of as few as two dominant correlation times is a
good approximation in many cases [285,286]. It was found
[287289] that thewater protonspinspinrelaxationcould
be described in terms of a population-weighted average of
two relaxation rates, characterized by motional correlation
times labeledbf (faster motion) andbs (slower motion):
1
1
2b
=
w
bf
1
2bf
+
w
bs
1
2bs
(24)
However, the case where more than one species of
water molecules each exhibits distinguishable motional
behavior and another case in which a single species of
water molecules exhibits anisotropic reorientation cannot
be distinguished.
3.1.4.
1
H T
1
relaxation affected by an exchange
A similar expression to that of Eq. (23) could be used
to describe T
1
relaxation in hydrogels. However, in the
T
1
experiment the population of polymer protons con-
tributing to the measured relaxation is not restricted to
exchangeable ones. As a result, the weighting factor p
c
,
usedinEq. (23) must be replacedby a parameter P
c
equal to
the ratioof polymer protons tothe total number of polymer
and water protons. This weighting factor does not satisfy
the condition of P
c
1, even for hydrogels of high equi-
librium water content. Hence, a more general equation is
required [290] to describe the spinlattice relaxation:
1
1
1
=
(1 P
c
)
1
1b
(z
ex
+1
1c
)
[(1 P
c
)z
ex
+1
1c
]
+
P
c
[1
1c
+z
ex
(1 P
c
)]
(25)
Here T
1b
and T
1c
are the spinlattice relaxation times
of the water proton pool and the polymer protons, respec-
tively.
Information on the molecular motions can be extracted
from the variable-temperature data for T
1
by rst invok-
ing a simple Bloembergen, Purcell and Pound (BPP) [40]
description for the intrinsic T
1
relaxation time correspond-
ing to each pool. The BPP theory assumes rapid molecular
motional averaging that is quite reasonable for hydrogels.
The following equation describes the dependence of T
1
on
the correlation time z
c
for the motion of molecules in a
particular pool:
1
1
1
= C
_
z
c
(1 +
2
0
z
2
c
)
+
4z
c
(1 +4
2
0
z
2
c
)
_
(26)
The constant C is a measure of the strength of interpro-
ton dipolar interactions. In order to estimate an activation
energy of the molecular motion, an Arrhenius law
z
c
= z
0
exp
_
u
R1
_
(27)
can be applied.
3.1.5.
1
H T
1
for polymers with no exchangeable protons
For gels that do not contain exchangeable polymer
protons, only through-space cross-relaxation between
the polymer and water protons can contribute to mag-
netization transfer. This process is most efcient in rigid
networks where neighboring protons have characteristic
short T
2
. The process is relatively non-efcient between
the polymer and water, since the approach of a water
proton to a polymer proton is limited by the length of H-
bonds, and the tendency for water protons to diffuse and
exchange rapidly among each other limits the strength
of the coupling. Therefore, in gels without exchangable
polymer protons, this contribution is negligible (P
c
=0
and z
ex
), so that T
1
=T
1b
. If the water molecules
exhibit anisotropic or multiple correlation time behav-
ior, similar to the T
2
case (Eq. (24)), it is assumed [288]
that:
1
1
1b
=
w
bf
1
1bf
+
w
bs
1
1bs
. (28)
3.2. NMR relaxometry on discrimination and
self-assembly of species with different mobility
In multiphase systems, each phase has its own char-
acteristic spinlattice and spinspin relaxation times that
correlate with molecular mobility. In order to sample the
wide range of T
1
and T
2
relaxation times, normally, the
saturation-recovery or inversion-recovery (for T
1
), and
CarrPurcellMeiboomGill (CPMG) or solid-echo pulse
sequences (for T
2
) are applied [26]. These experiments
result in obtaining the FIDs or magnetization decays fol-
lowed by their analysis through decomposing into two,
three or more signals decaying with various relaxation
times, based upon differences in the molecular mobil-
ity that reects structural heterogeneity and interactions
[291].
A three-state model for water in hydrogels was rst
proposed by Jhon and Andrade [169] based on the obser-
vation of structured water near the water/solid interfaces
and in natural macromolecular gels. Authors suggested
that water can behave dynamically and thermodynam-
ically as a part of the polymer chains when the water
molecules interact strongly with such specic sites as
the hydroxyl or ester groups, and classied this type of
water as the bound water. When the interaction between
the water molecules and the polymeric chains is weaker,
or when the water molecules are preferentially struc-
tured around the polymer network, intermediate water
is formed. The third type of water is free water where
interaction between the water and the polymer chains is
insignicant.
The decay in the transverse magnetization of water in
fully hydrated samples of PHEMA, poly(tetrahydrofurfuryl
methacrylate) (PTHFMA) and copolymers of HEMA and
THFMA was described by a tri-exponential function [292].
The short component of
1
H T
2
, T
2s
, was assigned to water
molecules that strongly interact with the polymer chains.
For PHEMA, the amount of water contributing to T
2s
,
expressed as a weight fraction of the dry mass of the poly-
mer (18%), agrees well with the value (20%) reported [168]
for bound water. The intermediate component, T
2i
, was
assigned to water residing in the pores of the sample [292].
The increased amount of water in the polymer network
effectively expands it to create a greater diffusive path
length, hence increasing the value of T
2i
. The long compo-
nent, T
2l
, was shown to arise fromwater residing in cracks
in the polymer network.
The proton spinspin relaxation in solids can be
described by Gaussian functions with T
2
=1215s. On the
other hand, the spinspin relaxation in semi-solids and
liquids can be described by Lorentz functions with a relax-
ation time of 16ms and above 10ms, respectively [293].
For T
2
decays in the region of 10200s, measurements of
T
2
are taken from the FID following a 90
rf pulse. Slower
T
2
decays can be obtained with the CPMG pulse sequence.
The 180
inverting pulses in the CPMG sequence effec-

tively refocus the spin dephasing due to magnetic eld
heterogeneity.
For the PNIPAm hydrogel beads, it was found with a
biexponential model for analysis of FID that the relaxation
times of water protons in the beads were in different time
ranges, suggesting a presence of several populations of
pores where water resides, and that each population of
pores had its distinguished average size [294]. The water
molecules residing within ner pores decay faster (short
T
2
) than those within larger (long T
2
). As the tempera-
ture increased, the pore size of the gel decreased gradually
while the relaxation times, T
1
and T
2
, showed different
patterns of change. It is believed that the
1
H relaxation
is affected by a direct temperature effect and an indi-
rect, temperature-related structural effect. The response
of the protons to these effects varied depending on their
location.
A temperature-dependent
1
H NMR relaxation study
was performed on chitosan and two crosslinked chi-
tosan networks with a different degree of crosslinking and
amount of added water [295]. An aperiodic saturation-
recovery sequence was applied for measuring T
1
, while a
CPMG sequence was used for measuring T
2
.
Due to the presence of fast and slow relaxing com-
ponents, deconvolution in the time domain of FIDs is
Fig. 22. The typical FID for polymer hydrogel (non-crosslinked chitosan
with [H
2
O]/[repetitive unit] =4.6): bottom an experimental FID; top
the result of simulation with three Gaussian components. The amount of
water, equivalent to the spin density, is obtained fromthe origin intercept
of each component [295]. Copyright permission fromAmerican Chemical
Society, 2001.
always necessary [296]. The shape of FID is t to a sum of
Gaussians
l = C
0
+
i
W
i
exp
_
t
2
C
2
i
_
, i = 2 or 3, (29)
where C
0
is the mean value of the noise, W
i
is the spin
density of the i component and G
i
is the width of the Gaus-
sian decay. The second moments M
II
i
of the FIDs were also
obtainedbyapplyingtherelationshipM
II
i
= 2]C
2
i
. Notethat
when more than one component is present their relative
amount is proportional to their spin density, which is cal-
culated as the origin intercept of each component of the
FID.
The analysis of T
1
relaxation data of hydrogels can be
cumbersome. In crosslinked networks with added water,
due to different T
2
relaxation times, two or three Gaussian
functions were used for obtaining the FID shape. As a mat-
ter of fact, in an aperiodic saturation-recovery multiblock
experiment, eachexperiment leads to anFIDobtained with
the proper time increment z
i
, with i =1. . ., N, where N is a
number of blocks. As shown in Fig. 22, at least two com-
ponents are clearly observed in each FID. Each FID can be
cut after a suitable initial delay [296]. In this way we can
obtain the T
1
value of the long T
2
relaxing component, T
1B
,
according to
l
Bi
= 8
0
+8 exp
_
z
i
1
1B
_
(30)
For obtaining the T
1
value of the short T
2
relaxing
component, T
1A
, a full best t of all other components
I
Ai
=I
Ti
I
Bi
, with i =1 . . . N is necessary.
A full deconvolution of FIDs allowed a plethora of infor-
mation [295]. For example, the dried samples of chitosan
and its crosslinked derivatives were partially rehydrated
and studied as a function of added water and tempera-
ture. The obtained relaxation data allowed estimation of
the number of water molecules in different solvation lay-
ers of polymer brils. Only about four water molecules per
repeat unit are tightly coordinated by chitosan while this
number increases for crosslinked chitosan and correlates
with the maximumswelling properties of the network.
The degree of crystallinity of the PVA cryogels was
determined by measuring the fraction of rigid protons
through
1
H FID measurements as a function of polymer
concentrationandnumber of freeze/thawcycles [119,120].
The FID in the PVA cryogels exhibits two components
characterized by a fast Gaussian decay with a relaxation
time on the order of 20s and a much longer expo-
nential decay. The former component, which corresponds
to a small number of PVA protons, is characteristic of
a rigid-lattice behavior, whereas the latter component
involves protons with different mobility. The rigid PVA
component is due to the hydrogel crystallinity. For low
numbers of freeze/thaw cycles, an increase in the num-
ber of freeze/thaw cycles or an increase in the polymer
concentration induces an increase in the percentage of
rigid protons. After six freeze/thaw cycles, the percentage
of rigid protons, which characterizes the degree of crys-
tallinity, tends to a limiting value of 7.4%. The degree of
crystallinity for aged PVA cryogels is higher than that of as-
preparedones. This observationwas explainedby a growth
of primary crystallites.
Calucci et al. showedthat theon-resonance
1
HFIDof dry
PAA-based crosslinked xerogels allowed at least two decay
curves to be distinguished [178180]. The experimental
decay can be reproduced using a linear combination of an
exponential, E(t), and a Pake, P(t), functions, describing a
slower and a faster magnetization decay, respectively
l(t) = P
E
(t) +P
P
P(t). (31)
The exponential function E(t) =exp(t/T
2
) is character-
ized by T
2
, whereas the Pake function can be written as
P(t) =
_
6
exp
_
2
t
2
2
_
_
_
cos t
t
_
C
_
6t
+
_
sin t
t
_
S
_
6t
_
,
(32)
where C and S are approximated Fresnell functions and =
3
2
h]4R
3
HH
. The Pake functionis therefore characterizedby
the parameters R
HH
, the distance betweentwo nearest pro-
tons, and , a parameter related to the dipolar interactions
between two non-nearest protons [178]. The best-t Pake
functiondescribing a very rigid component, contribute 78%
to the signal intensity. It is characterized by R
HH
of 1.78

A,
a distance close to that between protons in a methylene
group, andof 65kHz, corresponding to a T
2
value of about
20s. This indicates that spinspinrelaxationis mainlydue
to neighboring proton dipolar interactions modulated by
slowpolymer motions.
On the basis of
1
H MT, T
1
and T
2
relaxation analysis
in swollen PAA samples, while the water and non-
exchangeable polymer protons can be considered as two
separate pools, the water pool comprises water molecules
in different hydrogel environments and exchangeable
polymer protons, with negligibly small magnetic interac-
tions between the two pools [179]. Furthermore, the
1
H
data disclose information on the hydrogel structure as a
function of crosslinking degree. In particular, swollen PAA
samples with a low crosslink density are quite homoge-
neous with a relatively loose polymer network, in which
water diffuses fast between bulk and hydration environ-
ments. Onthe other hand, PAAhydrogel witha signicantly
higher crosslinkingdegreeshows aheterogeneous polymer
structure with regions characterized by different density
and wettability. In this case regions of high polymer den-
sity could formbeadlike structures which constitute nodes
from which polymer chains branch out giving rise to less
dense polymer regions embedding fast diffusing water. The
proposed beadlike structure [180] is expected to be more
rigid than the exible network observed at lower degrees
of crosslinking, and hence should exert higher constrain-
ing effects on the mobility of water molecules close to the
polymer surface.
The spinlattice relaxation times T
1
and T
1
allow
obtaining information on the degree of spatial heterogene-
ity in PAA hydrogel based on spin-diffusion [296] data. In
fact, the averaging effect of spindiffusionis complete inthe
T
1
but not in the T
1
time scale, allowing a rough estimate
of the homogeneous domains size based on Eq. (33)
-r
2
: = 6Dz, (33)
where spherical domains with radius r are assumed; D is
the diffusion coefcient, taken here equal to 10
16
m
2
/s, a
value valid for dense rigid proton systems [296], and z is
the time required for the magnetization exchange between
two spins in the Gaussian random walk model on which
Eq. (33) is based. Equating z with T
1
for PAA hydrogel, a
value for r 300

A was obtained [179]. On the other hand,
the best-t exponential function is characterized by a T
2
value of 46s. This result was conrmed by
13
C delayed
CP/MAS experiments where contributions from protons
with T
2
shorter than the experimental delay are sensibly
reduced; for PAA, a delay of 15s gave a spectrum with
intensity reduced by 75%.
The inversion-recovery decays of hydrated PAA sam-
ples obtained from the rst point of the FIDs could be
satisfactorily reproduced with a sum of two exponential
functions. The shortest T
1
time constant (T
1s
) is in all cases
on the order of 300ms, whereas the longest one (T
1l
)
increases with increasing water content from 800ms to
2.18s, whichis typical for the free water. Onthe other hand,
the inversion-recovery curves obtained using a point of the
Table 5
Activation energy of the molecular motion of polymer, E
p
, and water, E
w
,
in the pullulan hydrogel [298].
Concentration of pullulan, (wt.%) E
p
(kJ/mol) E
w
(kJ/mol)
27 21.5 17.2
35 24.3 24.1
41 28.3 28.3
FIDs at 800s, where the contribution of T
2s
is reduced,
could always be t to a monoexponential function with
T
1
=T
1l
. Hence, the obtained trend of proton populations
and T
1
and T
2
values [179] suggests a hierarchy of hydra-
tion: at low hydration levels water distributes around the
polymer chains until the hydrationlayer is saturated. Addi-
tional water tends to enlarge the polymer meshes until
the hydrogel is completely swollen and any further water
remains outside. The stepwise character of this process has
been previously observed in crosslinked hydrogels by ther-
mal measurements [172].
Three T
2
components of 10
2
, 0.11 and >10ms were
detected with the nonselective relaxation measurements
in hydrogels of the chemically crosslinked poly(acryloyl-l-
proline methyl ester) [297]. It was shown that the shortest
component belonged to the less mobile polymer protons,
while comparison with DSC results allowed assigning two
longer components to the non-freezing and freezing water,
respectively.
Okada et al. [298] detected with CPMG in the pullu-
lan(microbial polysaccharide of -1,6-linkedmaltotrioses)
hydrogels two relaxing species with different
1
H T
2
, which
they assignedto the inert polymer protons withthe shorter
T
2,1
and to the water protons with the longer T
2,2
. They
proved that hydration water and free water underwent
rapid exchange in hydrogels. Both T
2,1
and T
2,2
decreased
linearly with increasing 1/T, allowing estimation of activa-
tion energies of the molecular motion of polymer, E
p
, and
water, E
w
, in hydrogel. Both these energies become larger
with increase in the pullulan concentration, i.e., the mobil-
ity of pullulan chains as well as water molecules become
restricted at high concentration (Table 5). The mobility of
the labile protons of pullulan and hydration water is sup-
posed to be identical with the mobility of the inert protons,
because they move together when no exchange process
occurs. Thus, the difference betweenE
p
andE
w
is attributed
to the fractionand mobility of the free water. Withincrease
in the pullulan concentration the E
w
value becomes closer
to the E
p
value and E
w
=E
p
at 41% of pullulan. Under this
condition almost all the water molecules hydrate pullulan,
indicating that the hydration number per one glucose unit
is ca. 13.
The similar study based on relaxation measurements
and analysis of FIDs was performed, in order to reconstruct
phase diagrams, for lecithin/sorbitan tristearate (STS)
[299], and 12-hydroxistearic acid/canola oil organogels
[300,301]. In multiphase systems, each phase has its own
characteristic T
2
that correlates with molecular mobility.
The decays were tted with a number of Gaussian and
Lorentzian functions to determine the T
2
values for the dif-
ferent phases and their fractions. It was observed that oil
with 12% w/w STS showed about 11% solid phase, and 5%
semi-solid, while oil with 12% w/w lecithin showed only
about 6% semi-solid phase and no solid phase at all. The gel
with 12% w/w of STS and lecithin in oil at 50:50 ratio had
about 6%solid phase and 4%semi-solid phase inadditionto
the liquidphase. This indicatedthat the solidphase present
in the gel is due only to STS, while lecithin provides only
a semi-solid and a liquid phase. Fromthe obtained results,
the following model for this organogel was proposed [299].
The building blocks for the structure are provided by STS
crystals. Lecithin acts as a crystal type modier on the
STS, stimulating needle- or plate-shaped crystals, which
are more effective in building a network than other crys-
tals. The secondary function of lecithin is to promote the
formation of weak junctions between the crystals, forming
a network that entangles the oil in a rmgel.
Three main distributions were distinguished in canola
oil organogels with the T
2
values within the ranges of
5070ms, attributed to the immobilized or entrained oil,
80130 and 200500ms, attributed to the mobile liquid
oil in the temperature range studied [300,301]. With an
increase in the 12-hydroxistearic acid concentration, the
shorter T
2
values were observed. Therefore, an increase
in the 12-hydroxistearic acid concentration leads to an
increase in the connement of the liquid oil. With increas-
ing temperature, the immobilized oil at 5070ms becomes
part of the 80130ms range, and the 200500ms range
shifts to higher values. Furthermore, the highly immo-
bilized oil is almost nonexistent at 30
C. This suggests
that the liquid oil is not entrained efciently by the self-
assembled brillar network at 30
C as compared to 5
C.
This fact indicates that the supramolecular network is
more crystalline where the 12-hydroxistearic acid bers
aggregate into thick bundles with fewer inclusions in the
network.
The
1
H T
1
and T
2
relaxation measurements were per-
formed on poly(2-hydroxyethylmethacrylate-co-N-vinyl
pyrrolidone), poly(HEMA-co-VP), hydrogels with various
composition of HEMA/VP [302]. It was found that the
T
2
values were more sensitive to the polymer compo-
sition and the relative hydration than the T
1
values. A
short T
2
, detected in the homopolymeric PHEMA hydro-
gel, shows a high exchange rate of water molecules in the
polymer matrix, thus indicating that the water present
is interfacial water, which is highly mobile [244]. The
poly(HEMA-co-VP) hydrogels exhibited relatively longer
T
2
times as compared to PHEMA hydrogel with increase
in T
2
value observed along with increase in VP content in
copolymer and, hence, its hydrophilicity [302]. This obser-
vation suggested that the fraction of H-bounded water, in
poly(HEMA-co-VP) hydrogels is signicantly higher than
in the PHEMA hydrogel. The increase in content of bound
water results inthelower exchangerateof water molecules
within the meshes of the polymer matrix. This observation
is inagreement withrelaxationstudies of hydratedPHEMA,
poly(methacrylate-co-N-vinyl pyrrolidone) [170,173] and
hyaluron-based native and sulfated hydrogels [244]. It was
demonstrated that the T
1
and T
2
relaxation data correlate
with the diffusion kinetics of the hydrogels. Shorter relax-
ationtimes are indicative of Fickiandiffusionkinetics while
longer relaxation times are typical for the non-Fickian dif-
fusion [303,304].
The mobility of water in set yogurt during fermentation
was studied by
1
H NMR relaxometry [305]. The spinspin
relaxation was analyzed using two-exponential model,
resulting in two relaxation time constants T
21
and T
22
. Both
T
1
and T
2
relaxation times increased over the fermentation
period. This increasewas attributedtoanincreaseinmobil-
ity of water upon hydrogel formation. Water redistribution
within the gel matrix due to casein aggregation was shown
responsible for the changes in mobility.
Molecular dynamics of solvent was studied by
1
H
NMR relaxometry in organogel prepared from 1,2-O-(1-
ethylpropylidene)--d-glucofuranose and toluene [306].
It was shown that the toluene molecules are in the
fast-motion limit in both pure toluene and toluene
conned within organogel network in the studied
temperature range. The T
1
values of toluene in gel were
shorter than in pure toluene. This can be attributed to the
reduced mobility of the solvent which is entrapped within
the H-bond network formed by the gelator, increased
viscosity, and/or the possible interaction between the
toluene molecules and gelator aggregates which facilitate
a rapid dissipation of energy to the gel matrix. The surface-
dominated relaxation is reected in the distribution of the
measured relaxation times.
Sp eva cek and Suchoparek [307] detected polymer
solvent complexes in thermoreversible organogels of
syndio-poly(methyl methacrylate), s-PMMA, in bromoben-
zene and polyvinylchloride, PVC, in dibutyl phthalate or
diethyl oxalate measuring the selective and nonselective
1
HT
1
of the solvent. In all cases the relaxation decays were
monoexponential. While in pure solvent and in solutions
of atactic PMMA, where no associated structures exist, the
values of nonselective T
1
are always lower than selective
T
1
, as expected from the theory for
0
z
c
<1 [308]. On the
contrary, in s-PMMA gels the nonselective T
1
exceeded the
selective T
1
. Moreover, in all cases the nonselective T
1
val-
ues increased along with temperature, conrming that in
the studied s-PMMA gels the bulk solvent molecules are
on the high-temperature side of the T
1
curve, i.e., under
fast-motion conditions
0
z
c
<1. This conrms that the
reduction in selective T
1
values in s-PMMA gels occurs
because of formation of polymersolvent complexes. The
bound solvent molecules exhibit a slow-motion behavior
(
0
z
c
1) similar to that of associated polymer segments,
withfast exchange betweenboundandfree sites relative to
T
1
values (1s). Suchpolymersolvent complexes cancon-
tribute to the stabilization of the network and to promote
gelation of s-PMMA and PVC.
Combining the selective and nonselective proton spin
relaxation data obtained for PAAm/D
2
O/acetone gels
[309,310], both the physical and chemically crosslinked
PDEAAm hydrogels [103,104], and hydrogels of PIPAAm
or its copolymers [102,311314], a liquid-like behavior
was detected below the coilglobule transition. On the
other hand, motions with the correlation time of 2s,
which is characteristic for slowtumbling of globules, were
detected above the phase transition [103,104]. In the col-
lapsed state, the molecular mobility of both the water and
polymer occurred suppressed. The size of solid-like glob-
ules in the gels estimated by spin-diffusion measurements
using a GoldmanShen sequence equals 512nm [310].
As it was noticed yet by Sp eva cek [15], measurements of
only selective relaxation times for polymer protons lead
to the controversial assignment of motions either to the
dissolved low-molecular weight species [311], or to the
segments located on the surface of collapsed microgel
particles [312314]. In order to avoid these discrepan-
cies and misinterpretation, we could suggest utilizing a
combination of the solid-state CP/MAS and DD/MAS NMR
measurements [284] (Fig. 21) in this context.
The quite different range of motions was detected with
19
F T
1
measurements for uoromethyls in uoroalkyl-
ended poly(ethylene glycol) hydrogel, which reects
motions of the hydrogel network itself [315]. The variable-
temperature
19
F T
1
experiment conrmed the presence of
slow motions of these groups in the hydrogel network.
The T
1
values decrease along with an increase of tempera-
ture, which in turn demonstrates that T
1
increases with an
increase in correlation time. Therefore, the temperature-
dependent T
1
experiment reveals that the rotation of the
CCF
3
bond is in the slow-motion regime.
The fully relaxed dipolar decoupled
13
C (DD)/MAS spec-
trum of PVAiodine complex hydrogels indicated that
the hydrogels contain at least two components, a highly
mobile one and a broader component [316]. The for-
mer is assigned to the soluble or well water-swollen PVA
chains that are not closely associated with the PVAiodine
complexes, whereas the latter may be ascribed to the
aggregated PVA chains that are produced by the forma-
tion of the PVAiodine complexes. Furthermore,
13
C T
1
analyze reveals that the broader component is composed
of the highly restricted component assignable to PVA b-
rils assembled of the PVAiodine complexes and the less
mobile component. As for the former component, their
CH peak measured by the T
1
-ltering method was suc-
cessfully resolved into seven components. The statistical
analysis of the component intensities also revealed that
the probability f
a
for the formation of intramolecular H-
bonds in the successive two OH groups along each chain
and another probability f
t
of the trans conformation are,
respectively, as high as 0.86 and 0.88. This fact indicates
that the PVAiodine complexes shouldbe composedof PVA
segments with the trans-rich conformation.
3.3. NMR relaxometry on network structure and
crosslinking reactions
The NMR relaxation measurements are highly effec-
tive in study of kinetics and mechanism of gelation. The
1
H spinspin relaxation decays were applied to monitor
the network formation during the radiation-induced poly-
merization of NIPAAmin solution [317]. Before irradiation,
a single-exponential decay was observed with T
2l
1s.
After irradiation with doses in the range of 0.030.1kGy,
T
2l
decreased to 100ms, and a shorter component T
2s
appeared, of order of tens of milliseconds, indicating the
presence of network. At higher doses of irradiation, above
0.1kGy, the decay always consisted of two components, a
longer of the order of tens of milliseconds, and a shorter
of the order of milliseconds. The temperature dependence
of the relaxation time for samples irradiated with different
doses was in good agreement with swelling data.
Table 6
T
1
(
13
C) and T
1
(
1
H) values of the dry and hydrated PVA cryogels [322].
Peak Dry PVA cryogel Hydrated PVA cryogel
T
1
(
13
C), ms T
1
(
1
H), ms T
1
(
13
C), ms T
1
(
1
H), ms
CH II 4.40.4 3.00.2 6.10.4 3.90.4
CH III 4.30.2 2.60.1 6.60.4 3.40.2
CH
2
2.60.2 2.80.1 4.40.4 3.30.1
Assignment of signals was done according to Ref. [112].
Measurement of
1
H T
2
well above the glass transition
temperature allows determination of the crosslink density.
Anintroductionintothemethodis giveninRefs. [318321].
The presence of topological hindrances due to the junc-
tion points leads to an anisotropic motion of segments of
network chains. This results in a non-zero average of the
dipolar coupling and a residual dipolar magnetic interac-
tion of protons. The transverse magnetization decay M(t)
is composed of three contributions fromthe intercrosslink
chains, dangling ends and non-crosslinked chains (sol). By
tting the experimental FIDs, the parameters describing
the sol content and concentration of dangling ends as well
as the molecular weight of the network chains <M
c
> can be
calculated [318].
Dynamics of the polymeric domains in PVA and
PVA/lactyl chitosan (LC) cryogels was investigated with
1
H
DD/MAS and
13
C CP/MAS by measuring relaxation times
in the rotating frame,
1
H T
1
and
13
C T
1
, which are sen-
sitive to motions in the kHz range. The former averaged
over the intrinsic proton relaxation times are affected by
1
H spin diffusion and therefore depends on the neighbor-
ingspinreservoirs, whilethelatter gives informationonthe
local site-specic motions [322,323]. The
1
HT
1
and
13
CT
1
values of the dry and hydrated PVA gels are compared in
Table 6. The longer
1
H T
1
values observed in the hydrated
sample suggest that the spin reservoirs in the polymeric
matrix are characterized by a lower efciency of spin-
diffusion process on the kHz time scale. The common
1
H
T
1
of all peaks within the dry or hydrated sample together
with the observed lengthening of T
1
in the latter indicate
an increased mobility of polymer chains in the hydrated
gels relative to the dry gel. However, shortening of T
1
in
theatactic (a-)PVA/LCblendcomparedwiththat of thepure
a-PVA suggests that efciency of the spin-diffusion pro-
cess in the presence of LC increases. The opposite behavior
is observed in the syndiotactic (s-)PVA/LC blend as com-
pared to s-PVA. A similar result was obtained for
13
C T
1
of
the pure and blended PVA matrices, which in the hydrated
gels are signicantly longer than those of the dry samples,
witnessing a decreased efciency of the local site-specic
motions of methine and methylene moieties that govern
the dipoledipole relaxation mechanism[323].
The different
1
H T
1
values suggest that the spin
reservoirs of the two domains are spatially separated. It
was concluded that, since in the swollen domains T
1
of
methines are equal to those of the methylenes, the spin
reservoirs inthesedomains arehomogeneouslydistributed
andcharacterizedby similarly low-efcient relaxationpro-
cesses. On the contrary, in the PVA amorphous domains
T
1
values of CH peak II (71ppm) are longer than those
of peak III (65ppm), indicating that the spin reservoirs of
the methine moieties involved in intramolecular H-bonds
are spatially separated from those which are not. The
1
H
T
1
values of the hydrated gels have been measured with
contact times of 50s and 10ms to isolate the PVA reso-
nances of the methine and methylene moieties originating
frompolymeric domains with different mobility. At a con-
tact time of 50s, the methine and methylene signals from
the amorphous PVA domains are broad and characterized
by short T
1
, whereas at a contact time of 10ms, narrow
signals fromthe swollen polymeric domains, in which the
faster motions occur, are observed and show longer T
1
.
These results indicate that these motions occur in the fre-
quency range in which T
1
increases with a decreasing
correlation time.
Kinetics of gelation process in the course of mix-
ing aqueous solutions of polyions (copolymers of
N-(3-carboxy-2,4,6-triiodophenyl)-acrylamide or 2,4,6-
triiodophenyl acrylamide with sodium styrene sulfonate)
and poly(allylamine) was studied based on
1
H T
2
mea-
surements in order to estimate a change in chain mobility
[324]. The gradual increase in T
2
for water molecules
occluded in the polymer coils along with aging time
meant that formation of the polyion complexes is
accompanied by conformational changes of copolymer
chains. Simultaneously, T
2
values of the copolymer
chains decrease because of strengthening the polymer
bers.
1
H T
1
relaxation was measured in series of com-
mercially available contact lens hydrogels, made of
poly(N-vinylpyrrolidone-co-methyl methacrylate) and
poly(hydroxyethyl methacrylate-co-glycerol methacry-
late) by varying composition and equilibrium water
content [287,288]. These data were analyzed together
with
1
H T
2
relaxation data using a simple model involv-
ing magnetization transfer between water and polymer
protons. It was found that both data sets were generally
well described using common t parameters based on Eqs.
(23)(28), and the average water mobility in these materi-
als is governed by the equilibriumwater content, although
at a given content, slightly increased mobility was found
in the hydrogel materials that possess no exchangeable
protons on the polymer chains. The combination of T
1
and
T
2
measurements in a single model allows more accurate
quantication of water and polymer mobility and binding
in these hydrogels.
Degradation mechanisms of biodegradable photo-
polymerized poly(dl-lactide-co-glycolide) diacrylate net-
work were studied by
1
H solid-state NMR relaxometry
[325]. The network was degraded in phosphate buffer
solution of pH 7.4. A real-time
1
H NMR method pro-
vided information about the staged break down of the
network during continuous circulation of the buffer solu-
tion through the NMR tube. The degradation of network
proceeded in three stages: (i) extraction of a sol fraction
that causes substantial immobilization of the material, (ii)
scissions of network chains producing network defects
without formation of extractable products, and nally, (iii)
chain scissions that cause formation of a sol fraction and
complete degradation of the material. The T
2
relaxation
during rst two stages can be described with two compo-
nents whose characteristic times (1
rigid
2
and 1
elastic
2
) differ
by one order of magnitude. This means that two types
of polymer chains and/or chain fragments are present in
the sample. Values of 1
rigid
2
and 1
elastic
2
are typical for rigid
domains in multiphase polymers and viscoelastic network
chains, respectively. A third relaxation component with
very long decay (1
def
2
) appears during the last stage of
degradation. Value of 1
def
2
is typical for network defects
that are loosely attached to the network. The relative frac-
tion of these relaxation components represents the weight
fraction of the rigid fraction (up to 30%), the viscoelastic
network chains (up to 50%) and the highly degraded net-
work fragments in the residual hydrogel (up to 10%). This
heterogeneity couldbe due to heterogeneous spatial distri-
bution of network junctions in the initial network and/or
nanoscale phase separation of polyacrylate chains that
form multifunctional network junctions and poly(lactide-
co-glicolide) network chains.
Rather complicated segmental mobility occurring in
three different types of PNIPAAm hydrogels: normal
crosslinked gel (NG), comb-type grafted gel (GG), and
comb-type grafted gel with styrene-modied comb chains
(GG-st), was studied by variable-temperature measure-
ments of
1
H T
2
[100]. Decays for all three gels were
approximatelybiexponential. The decays of NGandGGgels
were almost identical, with short T
2
of 1213ms and long
T
2
of 130140ms. The overall magnetization decay of GG-
st gel (with a short T
2
of 11ms and a long T
2
of 97ms) was
more rapid than that of NG and GG gels. This indicates that
the GG-st gel networks in the equilibrium swelling state
are more rigid than NG and GG gels. Following the pre-
vious interpretation [326,327], the relaxation component
with a short T
2
value was ascribed to the restricted chain
segments, e.g., the segments near the crunodes, and the
component witha long T
2
corresponds to the highly mobile
segments.
It was found that at a temperature far below the LCST,
the T
2
decay of all three gels is still biexponential and
only weakly temperature-dependent up to 30
C. Above
30
C the shrinking of polymer segments occurs, which

results inthe decreasedsegmental mobilityof gel networks
and thus the decreased T
2
values. Above 32
C some poly-
mer segments become a dense solid-like globule which
cannot be detected in both the CPMG experiment and
the single pulse NMR experiment. The T
2
values mea-
sured above 32
C correspond only to the mobile polymer

segments (for example, at 34
C they reect the behav-

ior of about one-half of lone protons of the N-isopropyl
CH protons for GG gel, but at 35
C of small fraction
of these protons, while below 32
C T
2
values reect the
behavior of virtually all N-isopropyl CH protons). For GG
and GG-st gels, however, the T
2
relaxation is not longer
biexponential above 32
C. Instead, a well-resolved tri-

exponential decay curve, which corresponds to the rather
mobile non-collapsed structural units, is observed. The
three T
2
values obtained at 33
C are 4.5, 29 and 330ms

for GG gel and 3.2, 27 and 240ms for GG-st gel. Note that
the slowest decay found above 32
C in two comb-type
gels is absent in the NG gel. Such a slow decay compo-
nent was ascribed to the comb chains in GG and GG-st gels
which have free ends and higher mobility than backbone
chains.
The most interesting observation was done when tem-
perature increased from 33 to 34
C. The T
2
values of two
more rapid relaxation components for CH protons of the
N-isopropyl groups in two comb-type gels still decreased
substantially, but T
2
of the slowest relaxation component
increased abnormally. This implies an increase in mobility
of comb chains and a decrease in mobility of non-collapsed
backbone segments at 34
C, since the mobility of backbone

chains was mainly associated with two shorter T
2
compo-
nents and the mobility of comb chains characterized by
longest T
2
. For the GGgel, the longer T
2
component at 34
C
occupies 9.3% of all three relaxation components, which is
larger than that of the GG-st gel (3.5%). When tempera-
ture is further elevated to 35
C, the overall T
2
relaxation
of the GG gel seems to be biexponential, with the short
T
2
of 8.9ms (57%) and the long T
2
of 310ms (43%). Above
35
C, only weak NMR signal is observed (Fig. 7), indicat-

ing collapse of comb chains. This abnormal T
2
behavior
was interpreted as follows [100]. As temperature increased
from 33 to 35
C, some backbone segments of GG gel col-

lapse and become undetectable, while others shrink to
some extent which shortens T
2
of two more rapid relax-
ation components associated with main networks. During
this process, some parts of the comb chains are struc-
turally separated from (or protrude out of) the main gel
networks due to their higher mobility and thus become
more hydrated. As a result, the comb chains become freer
and have longer T
2
at 34
C than at 33
C. At 35
C, the
backbone networks shrink tightly, and their NMR sig-
nals become non-observable. In this case, the observable
signal is mainly contributed from the comb-type chains
which are still in mobile coil state. Therefore, the longer
T
2
component is expected to have larger fraction at 35
C.
This conclusion was supported by the result obtained for
GG-st gel.
Polymerization of tropoelastin at pH 11 yields a sta-
ble non-covalently linked hydrogel. Its formation is a
reversible process and likely to be due to hydropho-
bic entanglement of tropoelastin similar to the drying of
water-based latex paints. The
1
H T
1
values increased dra-
matically with increasing temperature whereas the
1
H T
2
values decreased [328]. More precisely, for T
1
the slow
component and its fraction, and for T
2
the fast com-
ponent and its fraction, both increased reversibly with
temperature. These observations are in sharp contrast to
the gained for another non-covalent hydrogel, gelatin. In
the absence of hydrophobic effects, T
2
increased expo-
nentially, and the opposite of the tropoelastin hydrogel
results. This was due to gelatin gel melting and becom-
ing a viscous solution above 30
C. It is interesting that
the gelatin hydrogel, in contrast to the gelatin solu-
tion, has reduced peak areas and relaxation decays that
were more easily tted with double exponential func-
tions, yielding a slower T
1
component as was seen for
tropoelastin.
3.4. Detection of slow mobility in gels with
one-dimensional exchange NMR experiments
Another way to detect slow processes occurred in
hydro-/organogels is an application of exchange NMR in
combination with the MAS technique. In such exper-
iments, the spin orientation of the sample nucleus is
detected before and after an adjustable mixing time z
m
.
For z
m
z
c
, there is no exchange of spin orientations
before and after z
m
, while for z
m
z
c
the exchange occurs
[329]. These requirements are met by the recently devel-
oped one-dimensional MAS exchange methods trODESSA
(time-reverse one-dimensional exchange spectroscopy by
side-band alteration) [330] and CODEX (center-band only
detection of exchange) [331]. In both methods, the length
of mixing time z
m
has to be synchronized with the rota-
tion period of the spinning sample. For z
m
z
c
, a normal
MAS spectrum is recorded, whereas if a dynamic process
has changed the spin orientation of the probe nucleus
during z
m
, a decay of the signal intensity in the MAS
spectrum is detected. For CODEX, additional 180
-pulses
are inserted in the evolution delays to amplify the fre-
quency encoding. In general, trODESSA is preferable if
the dynamic process involves large-angle reorientations,
the MAS can be set to rates smaller than the anisotropic
line widths and in all cases where the signal-to-noise is
a critical issue. In contrast, the CODEX experiment must
be applied if the dynamic process involves small-angle
reorientations only, or more detailed information about
the topology of the dynamic process (jump angles, etc.) is
wanted.
To determine dynamic parameters with trODESSA and
CODEX experiments, the line intensities vs. z
m
for all
resolved resonances in the MAS spectrumhave to be plot-
ted. For jump-like processes, these decays are proportional
to the correlation function of motion. Because many relax-
ation processes in the gel systems can be approximated by
stretched exponential KohlrauschWilliamsWatts func-
tion [332], the experimentally observed exchange decay is
described [329] as:
l(z
m
)
l(0)
= (1 u) +u exp
_
_
z
m
z
c
_
_
, (34)
where the parameter 0<1 is a measure for the width of
the z
c
distributionandthe parameter a characterizes topol-
ogy of the dynamic process. The latter can be extracted by
assuming motional models and performing numerical sim-
ulation. All experimental data I(z
m
) have tobe correctedfor
T
1
and spin diffusion [330].
13
C and
15
N solid-state NMR spectroscopy was used to
study the dynamic structure of a genetically engineered
multidomain [(AG)
3
PEG]
10
protein hydrogel that contains
two leucine-zipper domains and a central polyelectrolyte
domain [333].
13
C NMR spectra showed that on the ms
time scale the central domain is isotropically mobile while
the leucine-zipper domains are rigid. This suggests that
the central domain acts as the exible swelling agent of
the gel network while the terminal domains form inter-
molecular aggregates. Millisecond time scale motions in
the protein hydrogel were investigated using the
13
C-
detected
15
N CODEX technique [331,333]. The anisotropic
15
N chemical shift frequency is reintroduced under MAS
by a train of 180
pulses spaced at half a rotor period,

t
r
, apart, for N rotor periods. The
15
N chemical shift is
measured before and after a mixing period, t
m
, during
which reorientational motions occur. The changing molec-
ular orientations alter the
15
N frequency, thus modulating
the
15
N signal by cos(
1
2
), where
1
and
2
are the
time-averaged orientation-dependent frequencies before
andafter t
m
, respectively. Since cos(
1
2
) <1, reorienta-
tional motions reduce the
15
N signal intensities compared
to a reference spectrumacquired without the mixing time.
Thus, intensity differences between the exchange spec-
trum and the reference spectrum indicate the presence
or absence of motion. The CODEX-modulated
15
N sig-
nals were detected indirectly through
13
C by transferring
the
15
N magnetization to neighboring
13
C
sites using a
transferred-echo double-resonance (TEDOR) sequence.
13
C
detection provides both higher resolution and sensitivity
than
15
N.
While the leucine-zipper domains are rigid on the
s time scale, they are mobile on the ms time scale, as
manifested in the CODEX experiment. Fig. 23 shows the
13
C-detected
15
N CODEX spectra of protein for mixing
times of 50 (a) and 500ms (b), acquired with
15
N CSA
recoupling time of Nt
r
=727s. The reference spectra (S
0
)
at the two mixing times are nearly identical due to the
long
15
N T
1
relaxation time (7s). The exchange spectra (S)
show reduced intensities compared to those of the refer-
ence spectra, indicating that the
15
Nchemical shift tensors
have reoriented during t
m
. This reects reorientations of
molecular segments within as little as 50ms. The differ-
ence in intensities increased with the mixing time. Because
of
1
H
15
N cross-polarization, only the rigid fraction of the
protein was detected in the spectra, while the signals of the
mobile residues were suppressed. Note that only the
13
C
and
13
CO signals were detected, since these are directly
bonded to the amide
15
Nand were selected by the
15
N
13
C
TEDOR sequence.
The normalized CODEX intensities, S/S
0
, were plotted
as a function of t
m
in Fig. 24 for three resolved C
signals,
Ala, Glu, and Ser. Among these peaks, the Ala solid fraction
peak and the Ser peak have no contributions fromthe cen-
tral coil domain, while the Glu peak results mainly from
the helical domains but also contain small contributions
fromthe coil domain. Remarkable, the intensity decays for
all three peaks were well t by single-exponential func-
tions with the similar decay constants of 8112ms and
equilibrium values of 0.390.06. To gain more insight
into the nature of the leucine-zipper motions, the tem-
perature dependence of the exchange was examined. At
308K, the normalized exchange intensities (Fig. 24, circles)
yielded substantially shorter correlation times: 2816ms
for Ala, 4816ms for Glu, and 4622ms for Ser. The
reducedmotional correlationtimes at theelevatedtemper-
ature indicated that the leucine-zipper motion is thermally
activated.
Therefore, on the ms time scale, the leucine-zipper
domains are highly dynamic. The motion is a rigid-body
in nature with average amplitude of 50
at room temper-
ature. Several specic motional models are considered by
comparing simulated and experimental exchange intensi-
ties as a function of the recoupling time for
15
N chemical
shift anisotropy. The experimental data are consistent with
two of them[333]: a randomjump model and a non-axial
rotation model.
Fig. 23. Representative
13
C-detected
15
N-CODEX spectra of the
[(AG)
3
PEG]
10
protein hydrogel: (a) reference (S
0
), exchange (S), and
difference (S
0
S) spectra at t
m
=50ms, (b) reference (S
0
), exchange
(S), and difference (S
0
S) spectra at t
m
=500ms. Each spectrum was
acquired at 293K at a spinning speed of 5.5kHz. Nt
r
=727s [333].
Copyright permission fromAmerican Chemical Society, 2001.
3.5. PFG-NMR on self-diffusion of constituents of
hydro-/organogels
Pulse eld gradient NMR became a useful technique
for studying a self-diffusion of probe molecules in poly-
mer systems. Recently, high eld-gradient NMR system
with a maximum strength up to 2000G/cm and new
pulse sequences allow measuring the translational diffu-
sion coefcient at the order of 10
4
to 10
11
cm
2
/s in gel
systems [334]. A fewreviews and monographs in this eld
have yet appeared [334337]. In this section, some of the
most recent topics, especially characterization of the poly-
Fig. 24. Mixing-time (t
m
) dependence of the CODEX exchange intensity
S/S
0
for various C
sites: (a) Ala (55ppm), (b) Glu, and (c) Ser. Squares and
circles represent data acquired at 293 and 308K, respectively. Solid and
dashed lines are the respective best ts [333]. Copyright permission from
American Chemical Society, 2001.
mer gels and organogels by the diffusion experiments, will
be described.
3.5.1. Pulsed eld gradient spin-echo experiments
Detection of self-diffusion by means of pulsed eld
gradient spin-echo (PFGSE) NMR experiment has evolved
to become an important method in the characterization
of various colloid systems including hydro-/organogels
[42,43,334343]. For molecules undergoing Gaussian dif-
fusion, characterized by the self-diffusion coefcient D, the
echoamplitude I(,z,G,2z), where z denotes the time delay
between the 90
and 180
pulses, in such an experiment is

described [42] by the StejscalTanner equation:
l(, z, C, 2z) = l(0) exp(2zR
2
)
exp
_
(C)
2
_
z

3
_
D
_
, (35)
where I(0) is the signal intensity observed after the 90
pulse, denotes the duration of the magnetic eld gra-

dient pulse separated by a waiting period z between the
onsets of the two pulsed gradients which have the ampli-
tude G and are applied along the static magnetic eld B
0
(axis z). The echo attenuationgives informationonmolecu-
lar displacement along the gradient axis z that has occurred
during the waiting period z. Eq. (35) assumes that z is kept
constant. The normal experimental approach is to vary ,
and monitor the echo decay [338].
The stimulated spin-echo pulse gradient experiment
(PFG-STE) has the advantage that the time delay zis made
up of two parts, a shorter time z in which spinspin relax-
ation R
2
takes part and a longer time t in which spinlattice
relaxation R
1
occurs [43,340,341]. For gels in which R
2
is
large but R
1
is small, this method makes it possible to
extend the diffusion time zconsiderably. In this approach
a stimulated echo 90
pulse sequence is combined with

the application of two eld gradient pulses. Duration of
the gradient pulses is kept constant. Decay of the echo
intensity with increasing gradient strength G is a function
of the mean square shift of the observed molecules during
the waiting periodz[338]. The echoamplitude at time z +t
in the case of free diffusion is given [43] by Eq. (36):
l(, z, C, 2z, t) =
1
2
l(0) exp(tR
1
) exp(2zR
2
)
exp
_
(C)
2
_
z

3
_
D
_
(36)
Contribution from all the multicomponent diffusion
processes in the gel network can be considered. Normally,
for the hydro-/organogels, an increase in the diffusion time
enables the condition for restricted diffusion [340342] to
be satised. A convenient empirical approach to account
for the presence of distribution of diffusing species is
to use the stretched exponential, which is based on the
KohlrauschWilliamsWatts [332] equation:
l
j
(K) = exp[(KD
j
(upp)]
, (37)
where K=(G)
2
(z/3), and D
j
(app) represents the
apparent diffusion coefcient for component j. An average
inverse mean diffusion coefcient is here given [339] by
-D
j
(mean)
1
:=
1
1(
1
) -D
j
(app)
1
:, (38)
where 1(
1
) is the gamma function of
1
, and is a phe-
nomenological parameter that accounts for thedistribution
width of diffusion coefcients (0<1). For a monodis-
perse system equals to unity and Eq. (37) simplies to
the Eq. (35) [42].
In order to perform measurements of the multicom-
ponent diffusion in gel systems where the spectra of
components overlap for some reasons, PFG NMR was
extended to two dimensions. The procedure is termed
diffusion ordered spectroscopy (DOSY) [344,345], and
allows measuring the diffusion coefcients for compo-
nents by acquiring a series of one-dimensional spectra
with different amounts of diffusion weighting. The diffu-
sion coefcients are found by tting the variations in the
intensities of the peaks between the spectra. This informa-
tionis thenusedtogenerate a 2Drepresentationof the data
with the estimated diffusion coefcient along one axis and
the chemical shift along another.
3.5.2. Diffusion models in gels
Many theoretical models have been proposed to
describe the diffusion of solutes in polymer solutions and
gels, but in many cases, the models fail to describe the
diffusion of macromolecular solutes [44]. Diffusion mod-
els describing the solute diffusion in hydrogels can be
divided into a few categories according to the way they
interpret the retardation effect of gels on solute diffusion
(by obstruction, hydrodynamic interactions, reptation, and
entropic barriers to mention a few) [22,44,341].
The obstructionmodel assumes that the polymer chains
in the gel are static. This increases the path length between
two arbitrary points, the effect being a slower macroscopic
diffusion rate. Hydrodynamic theories imply frictional
interactions between all components of the system, the
most important interactions being assumed to be the ones
between the solute and the polymer [44]. De Gennes
introduced the idea of diffusion by reptation [22]. In
this case, a single polymer molecule is trapped inside a
homogeneous three-dimensional network of a crosslinked
polymeric gel andperforms wormlikedisplacements inside
tubular channels. In heterogeneous microstructures, the
entropic barrier transport model assumes that, if the diffu-
sant is a exible polymer, it may change its conguration,
which also allows passage through very crowded parts
of the gel from one cavity to the next [341]. In every
cavity, the number of possible congurations denes the
entropy of the diffusing polymer. The polymer tries to
increase own entropy by thermally activated jumps over
the intervening entropic barriers into less constrictive cav-
ities with enhanced congurational freedom. If the size
of the molecule strongly exceeds the size of the cavities,
diffusion can no longer be interpreted in terms of dis-
crete cavity-to-cavity jumps. Under these conditions, the
dynamics is described as a crossover from entropic bar-
rier transport to reptation, where the molecules reside in
several adjacent cavities and move upon expanding and
contracting.
Amsden [346] examined many diffusion models and
concluded that the one suggested by Cukier was the most
consistent diffusion model for homogeneous gels. Cukier
model [347] is a hydrodynamic scaling model describing
the diffusion coefcient as:
D = D
0
exp(k
c
r
s
3]4
), (39)
whereDandD
0
arethediffusioncoefcients withandwith-
out thepolymer, respectively, k
c
is anundenedparameter,
r
s
is the radius of the solute, and is the volume fraction
of the polymer. The model is based on the StokesEinstein
diffusion model, and it assumes that the solute molecules
are hardspheres that move at constant velocityina solvent.
The presence of the polymer chains increases the frictional
drag anddecreases the velocity of the solute or solvent near
the polymer chains. When the size of the solute and the
concentration of the polymer are known, the constant k
c
is
the only tting parameter.
Petit et al. [348] proposed a diffusion model in which
a hydrogel is considered as a statistical network with a
correlation length of . The solute diffusion occurs when
its molecule overcomes an energy barrier and jumps from
one cavity into the next one. The jump frequency over this
barrier, k, is dened in the formof the Arrhenius equation:
k = / exp
_

a
k
B
1
_
, (40)
where A is a frequency pre-factor, and E
a
the activation
energy of self-diffusion. Eq. (41) provides a description of
the diffusion coefcient based on this model:
D =
D
0
1 +uc
2v
(41)
where the constant a =D
0
/k
2
, c is the concentration of the
polymer, v is a constant for a given polymersolvent sys-
tem, whichdepends onthe quality of the solvent. The value
of k cannot be isolated because is unknown. However, the
activationenergy associated to the passage fromone cavity
to the other can be calculated with the variation of k
2
as
a function of the temperature. This model has been shown
[44] to provide a very good description of the diffusion of
solutes over a wide range of sizes and temperatures.
3.5.3. Self-diffusion of a solvent in hydro-/organogels
The translational diffusion of the entrapped water
molecules in various hydrogels was thoroughly studied by
means of PFG NMR [244,334,349364]. These investiga-
tions shed light on the microstructure of hydrogels, since
molecular transport is sensitive to their internal geometry
[357,358,361,363365].
The typical early measurements showed that in PHEMA
hydrogels, crosslinked with ethylene glycol dimethacry-
late [350], and in PNIPAAm microgels [334,365], the total
water phase diffuses as one homogeneous phase. The self-
diffusion coefcient was strongly dependent on the degree
of hydration of the gel, and practically no dependence on
the degree of crosslinking was detected.
The restricted diffusion of water was studied by PFGSE
NMR inthe gel network of three types of gellangumhydro-
gels, weak, true and brittle, which were obtained at various
concentrations of the gel-promoting potassiumor calcium
cations [351]. It was found that the topological structure
of the network, i.e., an average distance between junction
zones and permeability of the junction zone in each type
of gel is identical in both the K
+
- and Ca
2+
-briged gels and
the difference is only the gel-forming efciency of cations.
Water self-diffusion in aqueous model associative
polymer solutions, hydrophobically end-capped PEO,
C
12
EO
200
C
12
(AP9,) and C
12
EO
90
C
12
(AP4), has been stud-
ied with PFGSE and compared to the diffusion in the
non-modied PEO [359]. It was found that the diffusion
coefcient decreases monotonically with increasing poly-
mer concentration, giving D
i
/D
0
0.2 at 50wt.% (D
0
is the
neat water diffusioncoefcient), independently of polymer
molecular weight and modication. In further evalua-
tion of the data, the cell-diffusion model was used. The
cell-diffusion model [360] assumes that this decrease in
the diffusion coefcient is due to the combined effect of
obstruction and hydration of the polymer aggregates. This
model simulates a systemof xed particles, among which
the solvent molecules can diffuse. The key element of the
model is the widely used concept of dividing a macroscopic
system into smaller cells. Such an analysis suggested that
as water self-diffusion is mainly determined by the total
EO concentration in the solution, it is unaffected by the AP
molecules below C* of the parent PEO. On increasing the
temperature, water self-diffusion increases, following the
Arrhenius Eq. (40). The activation energy increases with
polymer content, and at 50wt.%, E
a
is 30kJ/mol indepen-
dently of polymer type. Almost the same value of 24kJ/mol
was obtained for diffusion of water in the curdlan (bac-
terial polysaccharide built of linear (13)--d-glucose
repeating units) gels [349]. Both these values exceed E
a
for
molecular diffusion in neat water, 17.6kJ/mol [366].
The similar result was obtainedfor the porous PVAcryo-
gels prepared by a freezethaw treatment of aqueous and
water-DMSO solutions [357,358]. The values of activation
energy E
a
for the self-diffusion of water in 10 and 15wt.%
cryogels were the same, 20.9 and 0.7kJ/mol above that for
the aqueous PVA solutions. This reveals that the morphol-
ogy of the continuous porous cryogel skeleton does not
depend on the initial PVAconcentration. The local viscosity
of water lling the cryogel pores differs from the viscos-
ity of the 10wt.% PVA solution. This is the more viscous
water, probably because of the strong H-bonds with the
PVA bers. It was shown that compartmentation makes
dramatic hindering of the water movement, e.g., the D
value for neat D
2
O at 25
C, 2.210
9
m
2
/s [362], is twice
as that for water in cryogels, 1.210
9
m
2
/s.
According to the electron micrographs [367], the PVA
cryogel consists of conning pores, but with interconnect-
ing channels which permit the solvent migration from a
pore to pore. A closely related behavior concerns the diffu-
sion of molecules in a labyrinth. The best characterization
of the self-diffusion type can be reached using depen-
dencies of the spin-echo attenuation I(q,z) against q
2
z,
where q=G/2 is a length of the inversed space vec-
tor q [343]. This vector has an analogy with the scattering
wave-number vector q. If the motion is inhomogeneous or
anisotropic, I(q,z) is a superposition of Gaussians, which
is uniquely determined by q
2
z, and of the nonlinear plot
of lnI(q,z) against q
2
z. For the restricted diffusion, where
the molecules experience modied diffusion arising from
physical barriers, the distribution of molecular displace-
ments is inuenced by the length scales of the systemand
the echo attenuation depends on zand q
2
separately and
not on q
2
z alone. Fig. 25 shows that in the PVA cryogels
the self-diffusion of water is anisotropic and sufciently
restricted[357]. Actually, one cansee that the self-diffusion
of water (and DMSO) in the cryogel is isotropic Brown-
ian only at 4
2
q
2
z<1.510
11
m
2
/s. At higher values of
4
2
q
2
zthe graphs for spin-echo attenuation become non-
linear and do not coincide with each other. The analogous
picture could be observed for other cryogels.
The effect of the number of freeze/thaw cycles and of a
drying/rehydration procedure on the water self-diffusion
Fig. 25. Dependence of the water proton spin-echo attenuation I on q
2
z
in cryogel of 10wt.%PVAin water: DMSO-d
6
=4:1 v/v at 20
C for zvalues
of 60ms diamonds, 100ms triangle, 200ms empty circles, 300ms
lled circles [357]. Copyright permission fromElsevier, 1999.
within the PVA cryogels was analyzed in detail with PFGSE
[368]. The samples on which just one cycle has been per-
formed exhibit the lower solvent mobility. With increasing
the number of cycles, the ratio D/D
0
, where D
0
is the dif-
fusion coefcient of neat water, increases and reaches a
maximum for twothree cycles, tending to a plateau. The
rst freeze/thaw cycle induces a microscopic phase sep-
aration in the system, accompanied by the formation of
crystallites in the polymer-rich phase. This means that
the polymer-poor phase is conned in regions embedded
in the network and constitutes a sort of interconnected
micropores [114]. Since the freezing procedure is quite
fast, the phase separation occurring during the rst cycle
may be not complete; i.e., it may not correspond to the
thermodynamic stability. Hence, the polymer-poor phase
contains more polymer than expected. On the other hand,
the polymer-richphase contains only fewsmall crystallites
so that, when the sample is thawed, the amorphous frac-
tionof the polymer-richphase canbe partially re-dissolved
in the pores. On repeating the freezethaw cycles, the
gel structure improves, and a further phase separation of
the polymer-poor phase within the pores occurs, forming
more dilute polymer-poor phase and a more concentrated
polymer-rich phase. Therefore, the increase of the D/D
0
value from 1 to 2 cycles may be due to two factors: the
decrease of the polymer concentration in the water solu-
tion swelling the pores and/or to the increase of the pore
size during the second freeze/thawcycle.
The structure of the crosslinked polyacrylamide gels
was studied using
1
H T
1
relaxation measurements and
PFG methods [369]. The diffusion measurements were
performed using the bipolar pulse pair (BPP) longitudi-
nal eddy-current delay (LED) stimulated echo sequence
[370] which effectively eliminates the effects of eddy
currents and sample-induced background gradients. The
data showed no effect of the crosslinker concentration on
the diffusion of water molecules. The OgstonMorris and
MackieMeares models [44] t the general trends observed
for water diffusion in gels. The diffusion coefcients from
the proposed volume averaging method [370] also t the
data, and this approach was able to account for the effects
of watergel interactions that are not accounted for in the
mentioned two theories. Contrary to the diffusion data,
T
1
relaxation measurements showed a signicant effect of
crosslinker amount on the relaxation of water in gels. The
effects of the crosslinker concentration were accounted for
by introducing the protonpartitioncoefcient betweenthe
bulk water and crosslinker phase. The partition coefcient
decreased signicantly as the crosslinker concentration
increased from5 to 10wt.%.
An interesting alternative is to measure diffusion
coefcients in the intense fringe-eld gradient of super-
conductive magnets [371], which turns out to be stable
and homogeneous. Further on, new modications to this
method were reported mainly to avoid the attenuation
terms due to relaxation times T
1
and T
2
[372]. This
method allows measurements of the time-dependent dif-
fusion coefcients D(t), reecting the existence of conned
geometries [373,374] within gels. D(t) for a crosslinked
polymer of sucrose and diepoxide monomers, hydrated at
18%and40%plottedagainst t
1/2
shows a linear dependence
[352]. The experimental points were tted by the relation:
D(t) = D
0
[1 /(D
0
t)
1]2
], (42)
whichis characteristic of a uiddiffusionina porous media
for short evolution times, and is attributed to slow dif-
fusion of molecules on the grain surface. The D(t) graphs
possess a linear dependence on t
1/2
except for the hydro-
gel hydrated at 96% where the graph is split in two zones,
which are interpreted as associated to free water and
structured water, respectively. The D(t) values converge
to a value of the order of 10
12
m
2
/s for t >2ms, which is
expected to be given by the hydrogel with more structured
water. The behavior of D(t) for the hydrogels at 18%and40%
suggests that almost all amount of water is structured. For
the hydrogel at 96% the bending at t
p
=1ms separates two
zones; onecorrespondingtoshort evolutiontimes t <t
p
and
the other for t >t
p
. In the rst region, the coefcients D(t)
remain almost constant indicating the existence of regions
in the sample with water molecules in a semiliquid state.
The time t
p
may represent an average mean diffusion time,
which allows estimation of an average pore size, or vol-
ume of trapped water, given by the average mean free path
d=(6D(t
p
)t
p
)
1/2
=2.510
6
m. It was suggested [352] that
a polymer, absorbing water to become a hydrogel, forms
different domains depending on the degree of hydration.
The more structured domains are linked to the chains by
H-bonds, the less structured domains will be formed by
water molecules in a semiliquid state.
The diffusion coefcients of N,N-dimethylformamide
(DMF) and tetrahydrofuran (THF) in Merrield network
polystyrene gels used as a solid-phase reaction eld have
been determined as a function of the degree of volume
swelling (Q) over the temperature range from 30 to 80
C
by means of the
1
HPFGSE NMR method [375]. It was found
that the D values for DMF in the network polystyrene gels
linearly increase with an increase of Q in the Q<3.2 range,
and a change of the D value for DMF in the Q>3.2 range
is smaller than that in the Q<3.2 range. The activation
energies of self-diffusion for DMF in polystyrene gels as
determined from the temperature dependence of D lin-
Table 7
The self-diffusion coefcients of D
2
O and mobile protons of the liquid
crystal hydrogels at 298K, parallel and perpendicular to the layer normal
[353].
Hydrogel D
mean
(D
2
O) 10
10
m
2
/s D
mean
(
1
H) 10
10
m
2
/s
Parallel Perpendicular Parallel Perpendicular
HG33d 0.840.02 7.090.07 0.720.02 4.200.12
HG65d 0.400.01 5.350.04 0.270.01 3.610.05
early decrease with an increase of Q in the Q<1.8 range,
and change of the E
a
value for DMF in the Q>1.8 range is
very small. The obtained data suggest that the Q depen-
dence of the D value for THF in the polystyrene gels is very
similar to that of DMF.
The water self-diffusion in the ordered lamellar liquid
crystal hydrogels based on 2,5-di-(4-{2-[2-(2-{2-[2-(2-
methoxyethoxy)ethoxy]ethoxy}ethoxy)ethoxy]ethoxy}
benzoic acid ester)benzoic acid 2-(2-methylacryloyloxy)
ethyl ester was measured in the direction parallel to the
layer normal and perpendicular to it, resulting in self-
diffusion components of D
||
and D
, respectively [353].
The linear dependence of the logarithmof the normalized
echo amplitude I/I
0
on the q
2
parameter signied that the
echo decay has a Gaussian shape, which means that there
were no restrictions to diffusion on the echo time scale.
The average diffusion coefcients of D
2
O and the mobile
protonated components are summarized in Table 7. The
measured values of D
||
and D
indicate that, on the one

hand, diffusion of water across the layers is possible and,
on the other hand, that the motion along the layers is not
free. The diffusivity along the layers is reduced by about
50% as compared to the free water, which can be explained
by the strong interaction with the ethylene oxide chains.
The anisotropy of D
2
O diffusion, D
/D
||
, was in the range
of 13.
The water diffusion coefcients in the hyaluronan-
based native and sulfated hydrogels crosslinked with
1,3-DAP or 1,6-DAE, (2.432.82) 10
9
m
2
/s, are lower
than in neat water at 310K [244]. The same conclusion
was done after study of PEG-diacrylate hydrogel. The rel-
ative diffusion coefcients for water in hydrogel and neat
water increased from 0.32 to 0.70 along with increasing
the molecular weight of PEG chains [354]. The echo decays
were monoexponential in all cases, thus excluding con-
tribution from other diffusing species and the presence
of barriers to the translational motion of water molecules
in the echo time of the PFGSE experiment. In fact, by the
Einstein equation <z
2
>=2Dt (where t =z/3 is the echo
time), the minimumamplitude for diffusional domains can
be derived (from38mfor hyaluronan/1,3-DAP to 41m
for hyaluronan/1,6-DAE), so that restricted diffusion under
these distances can be excluded [244].
Hence, the study of the solvent self-diffusion allows
obtaining information regarding the morphology of the
hydrogel matrix. To this effect, the apparent diffusion coef-
cient of water (D) is monitored as a function of total
diffusion time (z). In restricted geometries (such as hydro-
gel matrixes), D is unhindered at short diffusion times
and equals the diffusion coefcient of bulk water (D
free
).
However, as z increases, an increasing fraction of the
water molecules encounters network boundaries, thereby
restricting diffusion and lowering D to values less than
D
free
. At longz, all water molecules experienceboundaries,
resulting in a limiting value of D that may be correlated to
the root-mean-square (rms) end-to-end distance r of the
pore space in the hydrogel matrix via Eq. (33), and t
d
is the
value of z that approaches the limiting value of D [341].
The slope of the initial decay in D yields the surface area
to pore volume ratio (S/V
p
), and the magnitude of D at the
long-time plateau, which is indicative of the tortuosity of
the medium in which diffusion occurs. A rapid decay of D
in the short time regime indicates a larger S/V
p
, and lower
values of D at long times indicate greater tortuosity.
For the Ca
2+
crosslinkedAloe vera galacturonate (pectin)
hydrogels [355], the rms values of 14 and 13mwere cal-
culated by utilizing the individual D and t
d
values of the
0.2 and 0.6wt.% crosslinked systems, respectively. While
the calculated pore sizes of the two systems are similar,
the diffusion proles suggest subtle differences in hydro-
gel morphology. In comparison to 0.2wt.% hydrogels, the
0.6wt.% systems exhibit a sharper transition as a function
of z and lower values of D at long z, indicating greater
surface area and tortuosity.
The similar investigation was performed recently for
PNIPAAmhydrogels [364]. It was shownthat SAXS does not
provide information on connectivity of the pores. On the
other hand, PFG NMR can probe connectivity by measur-
ing the extent of connement of the water molecules. For
conned water, the spin-echo amplitude becomes inde-
pendent of zfor z>z. Eq. (33) provides a simple criterion
for determining the extent of connement of the water
molecules. For conned water with diffusion times z, z
depends only on the eld gradient G (i.e., on q) and is inde-
pendent of z. On the contrary, if the water is unconned
on the length scale 1/q, the decay for different z times is
a function of q
2
z. For PNIPAAm hydrogel, for example, if
connement of the water were dominant, the decay mea-
sured for z=90ms would appear to be 15 times slower
than that of z=6ms. The experimental data showed that
the effective size of pores was 38m, much larger than
that of 0.43mindicated by the SAXS measurements.
A logarithmic representation provided the distribution
of diffusioncoefcients g(D) of water intheswollengel. The
largest contribution fromthe water phase was centered at
a value of Dlower thaninbulkwater. The twosmaller peaks
of distribution were those of the water in the polymer-rich
phase. Thediffusionrateof thewater molecules varies from
one cavity to another and can be described by a Gaussian
distribution.
3.5.4. Self-diffusion of low- and high-molecular weight
compounds in hydro-/organogels
The low and high-molecular weight compounds intro-
duced into the hydro-/organogel serve as probes which
self-diffusion is capable to give a large amount of knowl-
edge on morphology of the polymer network and mobility
of brils.
The multicomponent self-diffusion of the PVA cryo-
gels prepared by a freezethaw treatment of the
water/oligooxyethylene glycol solutions of PVA was stud-
ied with the PFGSE method at various temperatures and
compared with the diffusion of such systems without gly-
col admixtures [376]. The evaluated activation energies,
E
a
, of the self-diffusion for the PVA chains showed that
entrapment of ethylene glycol, EG, and diethylene gly-
col, DEG, into cryogels made them more friable. These
glycols act as cryoprotectants. On the contrary, triethy-
lene glycol, TEG, and, especially, polyethylene glycol,
PEG-400, make cryogels rmer. The E
a
values for the self-
diffusion of oligooxyethylene glycols were in the range of
24.026.2kJ/mol exceeding those for the water molecules
in the same cryogels (22.423.8kJ/mol) and increased
along with the lengths of oligomers. These values are
also above those obtained for the PVA chains in cryogels
with EG, DEG, and TEG as well as without additives. This
fact is in conformity with the mechanism of the phase
separation of water and co-solvent by freezing the mul-
ticomponent system. It is supposed that the presence of
TEG and PEG-400 in cryogels leads to a decrease in dimen-
sions of the solvent-lled pores and the PVA laments like
that which was detected earlier for the DMSO containing
cryogels [357,358]. Such a compartmentation within cryo-
gels depends mainly on the dimensions of ice and polymer
microcrystallites formed by freezing the solution.
The similar diffusion measurements of PEGs with dif-
ferent molecular weights (108082,250g/mol) in whey
protein solutions and gels were carried out by Colsenet
et al. [377]. They revealedthat anincrease inthe size of PEG
resultedinareductioninits diffusioncoefcient. This effect
was more pronounced with increasing protein concen-
tration. Thermal aggregation of the whey protein caused
structural changes in the protein, leading to an increase in
inter-ber distances, enhancing the diffusion of all PEGs.
Similarly, a diffusion study of PEGs in curdlan gels [378]
showed a dependence on the curdlan concentration, the
size of PEG and the temperature. The diffusion coefcients
of polyethylene glycols, phosphate, trimetaphosphate,
alendronate, d-glucose-6-phosphate and cetylpyridinium
chloride micelles were examined. It was shown that the
reduction of relative diffusion coefcients is more pro-
nounced for larger solutes, a feature related to the mesh
size of the gels. The activation energy associated to the
self-diffusion was at the same order of magnitude as those
reported in diluted or semi-diluted gels. For a given solute,
there was no signicant difference between E
a
associated
to the diffusion of the solute free in aqueous solution
or incorporated in curdlan gels. This nding suggests the
lack of appreciable interactions between the solutes and
curdlan polymer. The diffusion data were analyzed accord-
ing to the diffusion models proposed by Cukier and Petit
[347,348]. On the contrary, from the self-diffusion mea-
surements for Boc-Gly and Boc-Phe in the polystyrene gels,
it was found that the low-molecular additives Boc-Gly and
Boc-Phe have the same diffusion coefcients as polymer
network because of intermolecular interactions between
these amino acids and the network [375].
The restricted self-diffusion of linear PEG macro-
molecules within crosslinked hydrogels of PMAAc, PNI-
PAAm, PDMAAm, PAAc, and copolymers poly(DMAAm-
co-AAc) and poly(stearylitaconamide-co-DMAAm) was
investigated by means of PFGSE in order to reveal the
effect of polymer/PEGcomplex formation [334,379]. It was
Table 8
Two-component diffusion of polystyrenes in the PMMA/toluene gels [383].
Concentration of PS, mg/mL D10
12
, m
2
/s Fraction of diffusion component
Fast Slow Fast Slow
6 1.4 0.18 0.55 0.45
10 2.0 0.44 0.77 0.23
20 2.7 1.00 0
50 0.8 1.00 0
shownthat inside the collapsedgel the PEGmolecules have
diffusionparameters likethoseof thenetworkchains [379].
This suggests the formation of an interpolymer complex.
In contrast, within the swollen gel the diffusion coefcient
of PEG molecules was independent of the diffusion time,
which indicates an absence of the interpolymer complex.
The internal structure of composite plum-pudding gels
made of responsive microgel particles inserted into a bulk
hydrogel (PNIPAAm microgel particles in a crosslinked
dimethylacrylamide matrix) was studied from the diffu-
sion of PEG probes through the network [380]. The biggest
decrease in the diffusion coefcient of the PEG probes
was obtained with the insertion into the bulk gel, with
only smaller decreases upon addition of the microgel
particles. This observation was explained by porosity of
microgel particles. Thus, the probe molecules could easily
diffusethroughthemicrogel particles, and, infact, diffusion
through the porous microgels may even be easier than dif-
fusion through the bulk gels, especially in the case of the
highly crosslinked microgel particles.
Diffusion coefcients of small organic solutes and large
micelles were as well slower in dextran gels than in D
2
O
[381]. For molecules with a molecular weight between
46 and 78, the self-diffusion coefcient in dextran gels
(20% w/w) corresponded, on average, to about 0.39 of the
values measured in bulk water. This hindered diffusion
was mainly associated with the obstacles created by the
polysaccharide segments. There was no evidence inthe dif-
fusion measurements of interaction between dextran and
solutes. The reduction of diffusion in dextran solutions and
gels was drastic in the case of micelles. For Triton X-100,
the self-diffusion coefcient in dextran gel was about 7%
of that observed in water. The values were, in fact, on the
same order of the dextran chain diffusion coefcient.
Other micelle-containing hydrogels were prepared by
drying and reswelling of the PVA hydrogel patches,
obtained by the freeze/thaw procedure, in decyltrimethy-
lammonium bromide (C
10
TAB) aqueous solutions. The
micelle diffusion coefcients were determined [382] from
the decay of the NMR peak of the methyl groups of C
10
TAB
as a functionof the square of the gradient strength, G
2
. Inall
cases, a monoexponential decay was observed, indicating
that the exchange of molecules between the local environ-
ment inwhichtheir mobility is different is fast withrespect
to the observation time (z=0.1s).
The two-component diffusion of probe polystyrenes
(PS) with M
w
from 4000 to 400,000 in the PMMA gels
swollen in CDCl
3
was measured by PFGSE with the diffus-
ing time zvaried [383385]. The diffusion coefcients for
the fast (D
fast
) and slow (D
slow
) diffusion components and
the corresponding fractions (f
fast
andf
slow
, respectively) are
shown in Table 8. Based on Eq. (33), the diffusion distances
for the fast diffusion component (r
fast
) were obtained to
be 1.03.3m, and the diffusion distances for the slow
diffusioncomponent (r
slow
) tobe0.40.9m. TheD
slow
val-
ues signicantly decrease with an increase in z, whereas
the D
fast
values decrease only slightly. This means that
the open network structure is so large (probably order of
10m) that PS chains see almost the same structure dur-
ingthediffusiontime. Ontheother hand, theformer may be
realized if the pertinent PS molecules are conned within
the dense network structure, the size of which is compa-
rable to the estimated diffusion distance (1m), during
the diffusion time z. Because f
fast
and f
slow
are almost con-
stant irrespective of the diffusion time z, it was concluded
that PS chains in the open and large-size and the dense and
small-sizenetworkstructures, whichcorrespondtothefast
and slow diffusion components, do not exchange within
the z range (from 40 to 500ms). This means that PS for
the fast diffusion component is also conned in the open
structures within the diffusion time. Moreover, the net-
work structure of PMMA gels prepared in toluene solutions
of PS with M
w
of 4000 and 400,000 are homogeneous and
inhomogeneous, respectively, within the z range. Thus,
the time-dependent PFGSE method using probe molecules
with different sizes is effective for structural analysis of gel
network.
In general, all the probe solutes introduced into the
gel networks are the good models for drug self-diffusion
when the gel systems are used as the drug carriers. The
lateral and rotational mobility of two drugs and one drug
mimic in photopolymerized poly(ethylene glycol) diacry-
late (PEGDA) networks were investigated by PFGSE as a
function of the crosslink density and temperature [354].
The network mesh size affects the lateral diffusivity for all
drugs, even if the mesh size is an order of magnitude larger
than the drug molecular size. The rotational motion is only
appreciably affected when the drug size and network mesh
size are of the same order of magnitude.
Regarding the drug model solute sodium salicylate
self-diffusivity in the hydrogels made of HPMC, HEC and
hydroxypropylcellulose (HPC) of various molecular mass
is not affected by the nature of the cellulose derivative
[386]. The observed exponential polymer weight fraction
w
P
dependence of the solute self-diffusion coefcient
D = D
0
exp(bw
P
) (43)
ascertains the validity of the free-volume theory, with the
diffusion coefcient D
0
for the pure solvent; b is a constant
indicative of the retardant effect of the polymer, which
equals 6.91, 6.30 and 7.36 for sodium salicylate in HPMC,
HEC and HPC hydrogels, respectively. Moreover, diffusion
takes place in the so-called bound water (which represents
about 40wt.% of the hydrogel). Solvent self-diffusivity in
the swollen polymer matrix is also compatible with the
free-volume theory in the lowpolymer fraction (up to 0.2),
but the steric hindrance of the polymer network is smaller
than that of the solute. Above this polymer fraction thresh-
old, water self-diffusion was found considerably restricted
because of interactions with the polymers. Finally, when
comparing the data for hydrogels containing 5 and 10%
w/w cellulose derivatives of high-viscosity with those of
low-viscosity grades, it was concluded that solute diffu-
sivity in the hydrogels is not signicantly affected by the
polymer molecular mass. It is dictated by the solvent vis-
cosity and the steric obstruction caused by the polymeric
network that lengthen the drug diffusional path, which is
the same at equal polymer weight fractions, regardless the
chain length.
Using bicontinuous microemulsions as templates opens
a new eld for the design of novel gel systems by
polymerization, but has signicant challenges due to
the very small composition and temperature windows
in which microemulsions are bicontinuous. Stubenrauch
et al. [387] had chosen a ternary base system water/n-
dodecane/Lutersol AO5 (n-alkyl polyglycol ether C
13/15
E
5
).
When monomer and crosslinker (NIPAAm and N,N
-
methylenebisacrylamide) were added to the water phase,
followed by addition of a gelator 12-hydroxyoctadecanoic
acid (12-HOA), gel was obtained. 12-HOA is known to self-
assemble into long brils that are connected via spatially
extended (pseudo)crystalline microdomains (nodes) [11].
The single brils have a diameter of 2030nm depend-
ing on the solvent, while the mesh sizes are in the range
of micrometers. On the other hand, the domain sizes of
a microemulsion are typically in the range of 1050nm.
The gelator proton signal could not be detected, because
the 12-HOA molecules formsolid brils. The characteristic
peak of the EO group allowed calculation of the surfactant
diffusion coefcient in the gelled microemulsion. The dif-
fusion coefcient for the oil molecules was obtained by
ttingtheareaof theCH
2
andCH
3
peaks withbiexponential
decay.
It is well known that the diffusion coefcients of oil and
water in a microemulsion are close to that of the respec-
tive continuous bulk phase. However, in a bicontinuous
microemulsion, free diffusion is possible in two dimen-
sions only and restricted in the third direction due to the
presence of the surfactant layer [339]. Thus, the theoreti-
cal diffusion coefcients of water and oil in a bicontinuous
microemulsion are 2/3 of the corresponding bulk values.
Obtaining the diffusion coefcients of water and oil in a
microemulsion, the connectivity of the solvents and there-
fore the type of microstructure can be deduced by plotting
the relative self-diffusion coefcients, D/D
0
, vs. the tuning
parameter of the system, which is the temperature in the
present case. The reference value D
0
is the bulk diffusion
coefcient of the solvent in the respective non-structured
system.
The water diffusion coefcient decreases while the oil
diffusioncoefcient increases withincreasing temperature
[387]. This means that inthe studiedtemperature range the
structure of the microemulsion changes from the water-
continuous tooil-continuous. Diffusionof NIPAAmcantake
place only in the aqueous phase, and thus, its decreased
diffusion coefcient reects the structural changes of the
microemulsion. The temperature effect on the NIPAAm
self-diffusion is slightly smaller than on the water self-
diffusion. The explanation is the fact that the increase
in temperature increases diffusion, which opposes the
decreaseinducedbythemicroemulsionstructural changes.
The surfactant diffusion is much slower than that of all
other components and slightly increases with increas-
ing temperature (by 20%). Assuming that all surfactant
molecules are adsorbed at the oilwater interface, i.e.,
neglecting the monomeric solubility of the surfactant in
the oil and aqueous phases, only lateral diffusion along the
interface can take place.
An increase in the temperature from the lower to the
upper phase boundary leads to the characteristic change
of the obstruction factors associated with a structural
change fromthe water-continuous to oil-continuous via a
bicontinuous microemulsion. The intersectionpoint occurs
at 35.7
C, and an obstruction factor is 0.325. At this

intersection the obstruction factors of the two solvents
are equal, which indicates the formation of a bicon-
tinuous structure [387]. Thus, the clear evidence that
the bicontinuous structure is maintained in the pres-
ence of the gelator, i.e., in the gelled microemulsion, was
obtained.
The effect of the k-carrageenan concentration on gel
microstructure and self-diffusion of probe molecules, PEG
and polyamideamine dendrimers has been determined
by TEM and PFGSE [388,389]. Gelation of k-carrageenan
is initiated upon cooling by a conformational transition
from random coils to double helices followed by aggre-
gation of the double helices into gel strands and network
formation.
Two dendrimer generations with signicant difference
in size were used: G2 and G6. The diffusion mecha-
nism for dendrimers is less ambiguous than that for PEG
because a dendrimer is less prone to change shape and
thereby diffusion mechanismas the surrounding structure
changes. Dendritic molecules are characterized by very lit-
tle polydispersity in contrast to PEG. It was shown that
the probe diffusion rate decreases with both increasing
the k-carrageenan concentration and PEG molecular mass
or dendrimer size. Small voids in the gel network gave
strongly reduced diffusion rate. It was established that the
deviations from Gaussian diffusion behavior come from
the two distinctly different diffusion coefcients of den-
drimers rather than microstructural heterogeneity effects
or a mean square displacement dependence on time that
is deviating from the dependence described by Eq. (33).
The average distance between gel strands was estimated
to be on the order of 10nm due to the ne-stranded gel
network and because the diffusion coefcient of the fast
diffusing dendrimers are in the magnitude of 10
11
m
2
/s,
rms displacement traveled by the G2 dendrimer is roughly
4m. Thus much larger than the distance between gel
strands (diffusive length typically 400 times the distance
between gel strands) and thereby much longer than any
conceivable open region made up by the k-carrageenan gel
network.
3.5.5. Self-diffusion of the hydro-/organogel network
The simplest and convincing application of the self-
diffusion measurements was recently demonstrated by
Brand et al. [390] for determination of T
SG
and hysteresis in
organogels of (trans-(1R,2R)-bis(undecylcarbonylamino)-
cyclohexane/toluene based on the temperature depen-
dence of ratio of diffusion coefcients for gelator and
solvent, which has a minimum at denite temperature of
T
SG
.
The model studies of polymer self-diffusion in the
physically crosslinked hydrogels were performed with the
complexes formed by gelatin and SDS or other alkyl sulfate
surfactants [391,392] and ethyl(hydroxyethyl)cellulose
(EHEC) and SDS [393] in water. One diffusion coefcient
is adequate to describe the behavior of the fractionated
gelatin at each composition, but two are necessary to
describe diffusion of the standard gelatin and EHEC. On the
other hand, the simplest model to understand the surfac-
tant diffusion data is a two-state model in which monomer
is in equilibrium with gelatin-bound micelles. The non-
complexed monomer diffuses at a rate that is expected
to be very similar to that in water, modied by a small
obstructioneffect due to the presence of gelatin. The micel-
lar species, on the other hand, are bound to the gelatin
or EHEC strands and therefore move together. Formation
of the micelle-mediated physical crosslinks between poly-
mer strands slows the polymer self-diffusion substantially
[391393]. It was concluded that maximal crosslinking
of gelatin occurs when there is about one micelle/strand.
Whenalarger number of micelles arepresent inthesystem,
crosslinking is less efcient because either fewer crosslinks
are formed, or they are each weaker, probably as a reec-
tion of increased electrostatic repulsion among the chains.
The transport dynamics in gel electrolytes based on
amphiphilic polymers was found to be faster than in gel
electrolytes based on corresponding non-amphiphilic
polymers [394]. The studied amphiphilic polymer
was polymethacrylate grafted with uorocarbon and
(CH
2
CH
2
O)
9
side chains, and the non-amphiphilic one was
a polymethacrylate with only (CH
2
CH
2
O)
9
side chains.
Self-diffusion coefcients of gel electrolytes with different
contents of lithiumbis(triuoromethylsulfonyl) imide salt
in -butyrolactone were determined by
1
H,
19
F, and
7
Li
PFGSE. The polymer diffusion coefcients showed that
the amphiphilic polymers seemed more intramolecularly
aggregated than intermolecularly. Moreover, the lithium
ion diffusion coefcient in the amphiphilic gel electrolytes
was found to be signicantly higher than that for cor-
responding gels based on the non-amphiphilic polymer.
The higher ethylene oxide content of the non-amphiphilic
polymer decreased the mobility of the lithiumions due to
cooperative coordination of lithiumions by ether oxygens
in comparison with -butyrolactone. The similar con-
clusions were made for aqueous solutions of hyaluronan
and hyaluronan functionalized with the steroid methyl-
prednisolone by means of CD, viscometry, rheology and
1
H PFGSE NMR [395]. Functionalization gives rise to very
compact species in solution, and competition between
intramolecular and intermolecular interactions depends
mainly on the amount of steroid subsistent and polymer
concentration. Samples with different molecular weights
have highlighted that competitive forces exist between
intramolecular interactions and internal stress according
to the intrinsic exibility of the polymer backbone. More-
over, samples with different concentrations have revealed
the competition between intermolecular interactions and
probability of different polymer chains to overlap.
Measurements for the non-covalently crosslinked
tropoelastin [328] showed that its diffusion coefcient
changed little between a highly concentrated tropoelastin
solution and the resulting hydrogel. The lower the concen-
tration, the higher was the estimate of D. In contrast, the
tropoelastin hydrogel formed a very stable non-covalent
network. The D value was similar for the aromatic and
aliphatic residues suggesting a comparable mobility of the
different side-chains within the hydrogel. The recurrent
nonlinearity of the StejskalTanner plots and the double
exponential decays for
1
H T
1
and T
2
measurements were
interpretedas being due tothe heterogeneityof the tropoe-
lastin sample.
Self-aggregation occurred with a critical minimum
tropoelastin concentration of 1mg/mL. This was estab-
lished by comparing the initial signal intensity with that
remaining fromthe supernatant after prolonged heating of
the sample. Thus, tropoelastin in the supernatant yielded
anincreased estimate of Dat higher temperature and a lack
of broadening of the
1
H NMR tropoelastin signals at LCST
(37
C). This was in contrast to tropoelastin hydrogel: the

solid seemed to rigidify (constant D at higher temperature
with broadening of resonances) when heated, suggest-
ing hydrophobically driven aggregation. The moderate
increase in T
1
observed for tropoelastin in the supernatant
is due to z
c
passing through the T
1
minimum, in the T
1
vs.
z
c
plot [40] when the temperature rose. This interpretation
is consistent with the observed higher D value and a lack
of broadening of the
1
H NMR spectrumat 37
C.
4. NMR spectroscopy on the morphology of
hydrogels and organogels
Clearly, the NMR spectroscopy is not a unique method
for study of morphology of hydro-/organogels. However,
there is a good opportunity to compare the data on mor-
phology acquired by NMR with those obtained with the
various diffraction (X-ray, electron, neutron) measure-
ments. Recently, NMR spectroscopy efciently competes
with diffractometry, and it is advantageous to gain these
complementing each other data together.
4.1. NOE measurements for study of hydro-/organogel
morphology
The presence of the NOE is indicative, normally, of the
existence of interpolymer complexes [396]. A solid-state
13
C{
1
H} NOEexperiment was performedtodetect thecom-
plexationbetweenPMAAanddeuteratedPEGincopolymer
blends and crosslinked networks [397]. For gels swollen
in acidic media at room temperature or at 37
C, strong
enhancements were detected in the
13
C resonance of the
PEG carbons. The NOE was generated due to energy trans-
fer between the rapidly rotating methyl protons and the
deuterated PEG carbons. In basic solutions, no NOE was
detected inthe PEG, as the complexes were dissociated and
the chains were separated in space. The NOEs observed for
the PEGcarbons for the chemicallycrosslinkedgels swollen
in acidic media were greater than those observed for the
physically crosslinked gels swollen under the same con-
ditions. This is due to the fact that a greater number of
complexes is formed in the chemically crosslinked gels.
However, the length scale of the interpolymer complexes
was shown to be similar for both gels.
In order to obtain information about the structure
of intermolecular aggregates of 1-butyl-3-[(3-
butylureido)phenylmethyl]urea organogel in CDCl
3
,
the
1
H NOESY experiments were carried out at different
temperatures and concentrations [72,73]. In the organogel
phase the cross-peaks between the phenyl and butyl
groups of the gelator molecule were detected. All the
NOEs in the aggregated state were negative, whereas
they were all positive in the non-aggregated state. This
indicates that the majority of the formed aggregates have a
molecular weight of at least 1500 or higher, which means
that these aggregates consist of at least six monomer
units. On the basis of the NOE contacts it was established
that there must be aggregates present with P2
1
, Pa or
P1 symmetry.
Various classes of peptide-based amphiphiles, which
are capable to form gels, have been reported up to date,
including amphiphilic peptides and peptides functional-
ized with alkyl tails on one or both termini [122125].
Amphiphilic peptides have been shown to form a vari-
ety of supramolecular-structure-like nanotapes, ribbons,
bers, andtwistedribbons, originatedfrom-sheet forma-
tion. They consisted of a linear hydrophobic tail coupled to
a peptide block that includes -sheet-forming segments,
and charged (amino groups in compound 3, or carboxyl
groups in compound 2) residues. Upon application of a
trigger such as a change in pH or ion concentration, these
charge-complementary molecules co-assemble in aqueous
solutionintobers containingamix of thetwocomponents
with-sheet H-bondingarrangements. Todemonstrateco-
assembly of the molecules within the bers,
1
H NOESY
was applied. Close contacts (<3

A) were observed between
the Glu-H
protons of 2 and the Lys-H
and Val-H
pro-
tons of 3, respectively. These results prove that the two
amphiphile molecules are co-assembled within the same
nanober.
Two additional types of intermolecular interactions,
namely, transient hydrophobic interactions between Phe
and methyl side chains and intermolecular -sheets
between the Asn-rich spacer regions were detected
recently with the solid-state
1
H NOESY in nucleoporin FG
(repeat domain) hydrogels [398]. The latter appear to be
the kinetically most stable structures. They are also a cen-
tral feature of neuronal inclusions formed by Asn/Gln-rich
amyloid and prion proteins.
The presence of ribbons in hydrogels from lipophilic
guanosine derivatives which are self-assembled into rib-
bonlike brils was detected with
1
H NOESY [126]. It was
shown that ribbon II (solution aggregate) is prevalent in
CDCl
3
, and is identied by a strong intermolecular inter-
action between guanidine NH
2
and ribose H(1
) and a less
intense cross-peak between NH
2
and ribose H(2
) protons.
The obtained data show that on passing from the crys-
tal to the solution aggregate, the conformation changes,
assuming a new anti arrangement in which the dihedral
angle y shifts to 175
. The solution conformation has

shorter distances for guanidine H(8) andH(1
) thanfor H(8)
and H(2
) protons because the cross-peaks relative to pro-

tons H(8) andH(1
) have larger intensity thanthose relative

to protons H(8) and H(2
). The passage from the crystal

to the solution conformation is probably connected to
the breaking of the weakest C OH(2) H-bond caused by
competition with the polar chloroform.
The self-assembly and packing of small peptidomimetic
cyclophanes in organic solvents was studied based on NOE
measurements [399]. It was shown that the self-assembly
of such compounds is very sensitive to small structural
changes such as the nature of the amino acid residue or
aromatic moiety, and the length of the aliphatic spacer.
Indeed, NOEs obtained by irradiation of the amide reso-
nance showedpositive enhancements at 50
Cbut negative
for the gels at 20
C. This is the result of the increased corre-

lation times upon aggregation and, accordingly, as pointed
out previously by Feringa and co-workers [72], the average
molecular weight is expected to be at least 1500, which in
this particular case is in accordance with the presence of
tetramers or larger aggregates in solution. By slow cool-
ing, at 50
C the molecules self-assemble in a crystalline

way that results in precipitation. The faster cooling down
to 20
C leads to aggregates long enough to drive the sys-

temtoward a columnar hexagonal packing that forms the
bers of the gel. These two packing modes have a dramatic
effect on the shapes observed by electron microscopy. In
one case, straight bers are observed, while in the other
case, helices arise.
To gain information about molecular arrangement in
the supramolecular aggregates it is valuable to know the
conformation of the molecules in solution. NOE experi-
ments were rather useful in identifying the conformations
with syn- or anti-type O CC
H dihedral angles. In
the anti-type conformations the amide hydrogen and the
proton attached to the chiral carbon atom are in close
proximity. As a result, the NOEs between these two
protons should be greater than those observed for the
syn-type conformations. The dihedral angles were around
3% for those in molecules with anti-type conformations
and less than 2% in the syn-type conformations. For com-
pounds which behave as organogelators in benzene, NOEs
higher than 3% were detected [399]. This strongly sug-
gests that anti-type dihedral angles are present in these
compounds.
A self-assembling hydrogel was formed from a mix-
ture of star-shaped eight-arm PEG end-modied with
-CD groups and the same star-shaped PEG end-modied
with cholesterol moieties [400].
1
H NOESY spectra
detected that the obtained gels are built due to for-
mation of -CD/cholesterol inclusion complexes. In the
NOESY spectrumof the PEG
8
20K-succinic anhydride (SA)-
-CD
7.4
/PEG
8
20K-SA-Chol
6.1
mixture in D
2
O (Fig. 26a),
cross-peaks from cholesterol protons (0.91.3ppm) and
both glucoside of -CD and PEG protons (3.9ppm) were
observed. Because the hydrophobic cholesterol moieties
and hydrophilic PEG chains are phase separated in the
Fig. 26.
1
H NOESY spectrum of a mixed PEG
8
20K-SA--CD
7,4
/PEG
8
20K-
SA-Chol
6,1
solution in D
2
O a. Cross-peaks of interest are indicated by
ovals. Hydrogel concept is based on inclusion complexes between choles-
terol (triangles) and -CD (cone-shaped structures) moieties coupled to
star-shaped 8-arm PEG b [400]. Copyright permission from American
Chemical Society, 2008.
aqueous environment, it was concluded that the marked
cross-peaks (enclosedintheovals) aretheresult of contacts
between cholesterol and glucoside of -CD protons in an
inclusion complex. The expected structure of this hydrogel
is shown in Fig. 26b. Hence, even such a qualitative consid-
eration of NOE contacts allows making conclusions about
the morphology of gel networks.
The structure of polymer networks formed through
hostguest complexation between -CD substituents of
6
A
-(1-(2-aminoethyl)amino)-6
A
-deoxy--CD substituted
PAAc (PAACD) and the adamantyl (AD) substituents
of 1-(2-aminoethyl)amidoadamantyl substituted PAAc
(PAAAD) was studied with
1
HNOESY [401]. Fig. 27a shows
cross-peaks (enclosed in the rectangle) arising from NOE
contacts between the protons of the -CD substituents
with the adamantyl H2H4 protons of the ADsubstituents.
The general pattern of the cross-peaks is consistent with
the dominant interaction occurring between the H3, H5,
and H6 annular protons of -CD and the AD H2H4 pro-
tons through the hostguest interpolymer crosslink shown
in Fig. 27b.
Hence, NOE measurements allow gaining the inti-
mate information on structure and morphology of a
hydro-/organogel network obtained in the course of self-
assembly.
Fig. 27.
1
H NOESY spectrumof a 0.5wt.% D
2
O solution in 3% substituted
PAA-CD and PAAAD with equimolar -CD and AD substituents (a). The
rectangle encloses the cross-peaks arising from interaction of the -CD
annular H3, H5, andH6protons withtheADH2-H4protons. (b) Illustrative
hostguest complex consistent with the spectrumin (a) [401]. Copyright
permission fromAmerican Chemical Society, 2010.
4.2. NMR diffusometry on morphology of
hydro-/organogels
Self-diffusion in porous materials like hydro-
/organogels is important by virtue its dependence on
molecular interactions or restrictions by physical barriers
within a pore [238,337,341,402,403]. The measurement
of the diffusive motion of uid or semi-uid molecules
by PFGSE allows extracting data on morphology of the
porous medium. The spin-echo amplitude obtained in a
PFGSE experiment can be understood in terms of a Greens
function or propagator P(r
0
,r,z) [42,340,342] of a diffusion
equation in the pore space which satises appropriate
boundary conditions
l =
_ _
(r
0
)P(r
0
, r, z) exp[i2q(r r
0
)]dr
0
dr, (44)
where (r
0
) is the equilibriumspin-density, z the period
betweentwo gradient pulses of magnitude and directionG,
and the reciprocal space vector q=G/2. The integral is
taken over all starting (r
0
) and nishing (r) positions of dif-
fusing molecule. Eq. (44) states that the echo attenuation is
given by the Fourier transformof the diffusion propagator
with respect to q. In the case of free diffusion, Eq. (44) leads
to
l = exp(4
2
q
2
Dz). (45)
Importantly, diffusion is measured along the direction
of the gradient. Eq. (45) states that the echo attenuation for
the free diffusion is given by a single exponential. Despite
the relative simplicity of the innitely short gradient pulse
approximation, apart from free isotropic diffusion, solu-
tions to Eq. (44) are only available for simple symmetrical
geometries, suchas betweenreecting planes separatedby
a distance a [340]:
l =
2[1 cos(2qu)]
(2qu)
2
+4(2qu)
2
n=1
exp
_
n
2
2
Dz
u
2
_
1 (1)
n
cos(2qu)
[(2qu)
2
(n)
2
]
2
. (46)
At long^, the secondterminEq. (46) disappears leaving
the long-time diffractive behavior with diffractive minima,
which arise from the rst term, appearing at q=n/a (n=1,
2, 3, . . .).
NMR diffusometry provides information concerning
uid saturation or the existence of uid layers adsorbed
at the solid matrix [337]. A special version of PFGSE
experiments (diffusion diffraction, q-space microscopy
[341,342]) has proven its capability to characterize con-
nected porous systems, which often has fractal properties.
Self-diffusion processes are sensitive to the dimensional
character of the path of diffusing particles due to the
microstructure. For the free diffusion of uid with D
f
, the
mean square distance from the origin at t >0 is governed
by Eq. (33). For the restricted diffusion it holds [337]
-r
2
:D
f
t
2](2+0)
. (47)
The effective dimensionality of the randomwalk, 0, van-
ishes for Brownian motion. For sufciently long times, the
mean square distance <r
2
> becomes a linear function of
time
-r
2
:D
r
t. (48)
The self-diffusion coefcient D
r
for restricted diffusion
is also called apparent diffusion coefcient for long times
(see Eq. (33)). Besides D
f
, D
r
, and anaverage radius of pores,
further parameters characterizing the structure of the pore
system can be derived as a function of z: the tortuosity
T =D
f
/D
r
or [(1)D
f
/D
r
]
1/2
(where is mean porosity),
the surface-to-pore volume ratio S/V, the pore-size distri-
bution, characteristic lengths, and the permeability [337].
The molecular translation displacement probability
characterizes an average pathway of liquid molecules
imbibed in a microporous medium. Under the narrowPFG
approximation, displacement proles can be obtained by
Fourier transform of the spin-echo attenuation proles
measuredas a functionof the amplitude of the pulsedmag-
netic eld gradient (the so-called q-space NMR imaging).
For interconnectedpore systems inhydro-/organogels, this
approach can yield not only the diffusion coefcients but
also the mean pore spacing and mean pore size [404,405].
The molecular displacement proles of water molecules
in cellulose ber samples with different water contents
Fig. 28. PFGSE echo attenuation proles as a function of q at six different
diffusion times zfor a cellulose ber sample with 81.7wt.%water a, and
b average diffusion propagator of water in the sample at these diffusion
times [406]. Copyright permissionfromAmericanChemical Society, 1997.
were measured as a function of the diffusion observation
time z=51200ms [406]. The time course of the transla-
tional displacement proles indicates the presence of both
the free-diffusing and restricted water inthe samples over-
saturated with water. Water molecules exhibit essentially
restricted diffusion behavior when the diffusion observa-
tion time z is sufciently long. The anisotropy of water
diffusion in cellulose bers was also investigated by using
atetrahedral gradient combinationpatternconsistingof six
different combinations of simultaneously applied orthog-
onal gradients (x, y, and z gradients).
Fig. 28a shows the spin-echo attenuationproles at var-
ious diffusion times zfor an oversaturated cellulose ber
sample with a water content of 81.7wt.%. The echo ampli-
tudes are presented as a function of the variable q. Fig. 28b
shows the Fourier transformof these proles (an averaged
propagator <P(R,z)>) for a distance of R=(r
0
r) <60m
at different diffusion times. It is seen that at short diffu-
sion times the average translational displacement prole is
nearly Gaussian, indicating most of the water in the sam-
ple undergoes free self-diffusion. As the diffusion time was
increased, the translational displacement proles deviate
from the Gaussian distribution. Compared with free dif-
fusing water, the averaged translational displacement for
water in sample is higher at short distance but lower at
larger displacement. Since the permeability of the cellulose
ber is negligible during the diffusion time used, the data
presented in Fig. 28b suggest that there might be both free
diffusing water and restricted water in the ber sample.
It was mentioned yet that in the PVA cryogels the
self-diffusion of both the polymer chains and the aque-
ous molecules is anisotropic and very much restricted
[357,358]. Despite the fact that both the water and the
PVA chains have a restricted motion, we were capable
to observe the interference effects in a q-space for the
PVA chains only. For diffusion in a pore-glass composed
of interconnecting spherical pores of diameter b, the rst
peak maximum occurs at q
max
=b
1
[343]. This, the so-
called pore-hopping interference effect, is different in
its origin from that produced by a diffusion of spins that
are restricted to move inside single, non-interconnected
compartments [342]. The former effect is analogous to
the optical two-slit interference effect, while the latter
corresponds to single-slit diffraction [343]. Asimple esti-
mation according to the observed two q
max
values gives
two characteristic lengths b for a cryogel of 10wt.% PVA in
D
2
O: 1000 and 600nm[357].
For the estimation of geometric parameters of the
water-lled pores and the PVA network of cryogels a
pore-hopping formalism worked out by Callaghan et al.
[404,405] was applied. The pore-hopping theory is based
on the assumption that diffusion between pores is much
slower than within a single pore. In order to t the data
using the pore-hopping theory, Eq. (49), allowing for some
deviation in the pore spacing b, was used. Provided <b
and a Gaussian distribution of the standard deviation in
b, spin-echo attenuation is
l(q, z) = |S
0
(q)|
2
exp
__
6D
eff
z
(b
2
+3
2
)
_
_
1 exp(2
2
q
2
2
) sin(2qb)
2qb
__
. (49)
The structure factor S
0
(q) for the spherical
pores/polymer drops with an average diameter a is
S
0
(q) =
3[2qu cos(2qu) sin(2qu)]
(2qu)
3
. (50)
Fig. 29 shows the data for various ztogether with the-
oretical curve for the maximal z (400ms) obtained using
Eq. (49) in a least-squares t the parameters a, b, , and D
eff
[357,358]. Here we have assumed for simplication that
|S
0
(q)|
2
arises from Eq. (50) for spherical pores. These ts
for restricted diffusion of water and PVA chains yield the
structural parameters shown in Table 9. The PVA signal
attenuation allows estimation of the geometric parame-
Fig. 29. Dependence of the PVA proton spin-echo attenuations against
q and z in cryogels of 10wt.% PVA in D
2
O, 20
C, =5ms, for z values

of 60ms (lled circles), 100ms (empty circles), 200ms (lled triangles),
300ms (empty triangles), 400ms (squares). The solid line is the nonlinear
least-squares t using Eqs. (49) and(50), z=400ms. The obtainedparam-
eters are shown in Table 9 [357]. Copyright permission from Elsevier,
1999.
ters of the structural elements of the cryogel bers. Table 9
shows that these bers in a cryogel of 10wt.% PVA in D
2
O
have diameter a =60nmand length b=570nm. The length
of bers coincides with the second characteristic length
b
2
=600nmdetermined fromthe second diffraction maxi-
mum.
Fittingtheattenuationfor thewater signal at z=300ms
(Fig. 25) to Eqs. (49) and (50) detects large pores in
cryogel of 10wt.% PVA in D
2
O with an average diameter
a =1200nm (Table 9). This coincides with the scanning
microscopy data showing that the pore sizes are about
1000nm in diameter [367]. These pores are lled with
water and interconnected by channels having an average
length b=2400nm(Table 9).
Chui et al. [407] proposed a method based on measure-
ments of spin relaxation times in hydrogels to obtain pore
radius distribution proles. Data for spinlattice relax-
ation times of the water in the gel were interpreted by
usingthemagnetization-diffusion equationthat has been
applied previously for similar measurements of porosity
in glass, sandstones and so on. To account for the brous
structure of hydrogels, a ber-cell model was introduced.
This model allows interpreting the mono- and multiex-
ponential relaxation behaviors for hydrogels according to
the competition between diffusion and relaxation of pro-
ton spins. It was demonstrated that agar, agarose and
polyacrylamide hydrogels satisfy the criterion for fast dif-
Table 9
Structural parameters innmobtainedbyttingPFGSEdatatoEqs. (49) and
(50) for various cryogels of 10wt.% PVA, 20
C [357]. See an explanation

in the text.
Cryogel Water
a
PVA
b
a b a b
In D
2
O 1200 2400 1100 60 570 200
In D
2
O:DMSO-d
6
=4:1 1000 1000 800 60 250 220
In D
2
O:DMSO-d
6
=2:1 870 900 600 50 280 230
a
z=300ms, =1ms.
b
z=600ms, =6ms.
fusion, and the pore-size distributions for these materials
were calculated. Recently, a 2D NMR proton relaxometry
and diffusometry were used to compare the porosity of
hydroxyethyl methacrylate-based hydrogels used in con-
ventional contact lenses with silicone hydrogels developed
for continuous-wear contact lenses [408]. It was shown
that both types of hydrogel have a connected network of
nanopores, but the silicone hydrogels additionally contain
pores of the micrometer scale that enhance their perme-
ability.
4.3. NMR cryoporometry
The method for estimation of an average size of pores
and channels within gel structures, based on NMR dif-
fusometry [340342], was discussed above; besides, the
distributions in these sizes are also required in order to
characterize morphology of gels. The recently developed
approach called NMR cryoporometry allows perform-
ing determination of this pore-size distribution (PSD). The
perfect review describing the last achievements with this
approach was recently published [409].
NMR cryoporometry provides a quantitative analysis of
pore size in the nanometer range, a regime where imag-
ing techniques have yet to achieve sufcient resolution to
access. With respect to the importance of porosity for the
development of both micro- and nanosized biodegradable
drug-depot formulations, NMR cryoporometry is based on
the fact that the spinspin relaxation time for the liq-
uid NMR signal is several orders of magnitude longer
than that for solid. By choosing water as a probe liquid,
another advantage of this approach becomes the possibil-
ity to determine pore-size distributions, which offers the
opportunity to monitor the change in size distribution as a
function of time.
The general idea of NMR cryoporometry is to detect
the shift of phase transition temperatures for a solvent
that is conned in pores. This shift can be interpreted in
terms of pore geometry and provides information about
pore sizes and their distribution and, in favorable cases,
about pore shape. The solvent transition is solid-to-liquid,
or vice versa, depending on the direction of the temper-
ature change either melting or freezing. The underlying
reason for a shift in the melting and freezing points is the
cost of creating a new interface with a non-zero surface
tension [409]. Normally, NMR cryoporometry relies on one
particular recapitulation of the GibbsThomson equation
developed for the case of cylindrical pores with radius r.
Jackson and McKenna [410] expressed the shift of the pore
melting/freezing point T
m
as
z1
m
= 1
m
1
0
=
k
r
, (51)
where k is a material constant that depends on the molar
volume v, the solidliquid surface energy, the latent heat of
melting (freezing) ^H and the bulk solvent melting point
T
0
.
Typically, the emergence/disappearance of the liquid
signal upon increasing/decreasing temperature in a spin-
echo experiment is detected. In the case of distribution
of pore sizes, and, hence, a related distribution of melting
points according to Eq. (51), the resulting NMR signal of
intensity I varies with temperature. Its shape reects the
pore-size distribution. By assuming that I(T) V(T), where
V(T) is the pore volume that contains molten solvent at a
given temperature T, one can obtain a pore-size distribu-
tion p(r):
p(r) =
k
r
2
dl(1)
d1
. (52)
Note that this pore-size distribution is, in the absence
of absolute scaling between I(T) and V(T), a relative one. It
is, though, simple to obtain an absolute pore-size distribu-
tion and total porosity (or specic pore volume) by proper
procedures of scaling [409].
Up to date there are still only a few publications
concerning determination of the pore-size distribu-
tion in hydrogels [411413]. The nanoporous structure
of collagen-glycosaminoglycan (CG), polymethylsiloxane
(PMS) and gelatinized starch hydrogels was studied using
1
H NMR cryoporometry and thermally stimulated depo-
larisation current method with layer-by-layer freezing-out
of bulk and interfacial water. The depression of the freez-
ing point for the conned water is related to the size
of the nanopore. Changes in the Gibbs free energy of
the unfrozen interfacial water are related to the amount
of bound water in the hydrogel matrix and to the re-
arrangement of the 3Dnetwork structure of a polymer. The
use of the GibbsThomson relation for the freezing point
depression allowed calculating [411]: (i) the thermody-
namic parameters of interfacial water weakly and strongly
bounded to polymer molecules, (ii) size distributions of
pores lled by structured water, and (iii) surface area and
volume of micro-, meso- and macropores.
Re-hydration of the CG gel did not restore the amount
of bound water (initially up to 90wt.%). This effect was
explained by structural changes in the 3Dnetwork of colla-
gen molecules. Freeze-drying of the initial hydrogel causes
theformationof additional collagencollagenbonds, which
are not destroyed in the re-hydration step, and this leads
to diminution of the amount of the bound water and a
decrease in the absolute value of interfacial energy mea-
sured by
1
H NMR [412]. Estimation of the temperature
dependence of the size andvolume of nanopores lledwith
unfrozen water showed that short-range order in freeze-
dried CG gels changes insignicantly in comparison with
long-distance order. Three types of structured water were
observed in the PMS xerogel (adsorbed water of 3wt.%).
These structures, characterized by the
1
H chemical shifts
of 1.7, 3.7, and 5.0ppm, corresponded to weakly associ-
ated but strongly bound water and to strongly associated
but weakly or strongly bound waters, respectively [413].
Pore-size distributions obtained with NMR cryoporometry
suggested that PMS is a mesoporousmacroporous mate-
rial with the textural porosity caused by voids between
primary particles forming aggregates.
4.4.
129
Xe NMR in the porosity studies
Xenon is a hydrophobic, chemically inert gas. It pos-
sesses a highly polarizable electron cloud that makes the
129
Xe NMR chemical shift very sensitive to the surround-
Table 10
129
Xe NMR chemical shifts and line widths in the presence of dry and
hydrated atactic (a) and syndiotactic (s) PVA, PVA/lactyl chitosan cryogels
and lactyl chitosan (LC) [323].
Polymer Dry gels Hydrated gels
, ppm ^
1/2
, Hz , ppm ^
1/2
, Hz
LC 1.8 48 187.9 46
188.3 22
a-PVA 3.9 86 187.3 85
7.6 289 189.6 194
196.2 152
s-PVA 4.8 86 189.1 81
17.7 1040 190.0 128
200.6 600
a-PVA/LC 2.2 115 188.5 27
3.8 226
s-PVA/LC 2.4 95 187.8 30
10.0 390 188.2 56
ings. It has been shown that
129
Xe NMR is a powerful
tool for investigating the morphology and local environ-
ments of complex molecular systems, such as proteins,
biological membranes, anda variety of polymeric materials
[414,415].
The hydrophobic character of the polymeric domains of
the PVA and PVA/lactosilated chitosan (LC) gels was inves-
tigated with
129
Xe NMR [323]. Both the dry a-PVA and
s-PVA samples exhibited two
129
Xe signals shifted down-
eld fromthe signal of pure gaseous xenon, characterized
by quite different line widths, which are in the slow-to-
intermediate exchange limit on the
129
Xe NMR timescale
(Table 10). This result indicated that xenon is trapped
within the polymeric matrix of dry a-PVA and s-PVA. In
particular, the broader resonance of s-PVA is characterized
by a larger downeld shift and line width than in a-PVA.
This suggests that the arrangement of the hydrophobic
environments in which xenon is adsorbed are signicantly
different in the a-PVA and s-PVA domains; furthermore,
the much larger line width observed in s-PVA indicates the
existence of more inhomogeneous environments and/or a
strong reduction of the T
2
relaxation time.
Comparison of the broadest lines in the blends with
the corresponding ones in the pure PVA indicates that
in the former, LC rearranges the polymeric matrix, giving
rise to microdomains magnetically different fromthose of
the pure PVA characterized by a higher homogeneity and
by longer T
2
relaxation times. Signicant changes in the
polymeric matrix of the blends are also suggested by the
broadening of the high eld signals (Table 10). Indeed, s-
PVA/LChas a denser matrixthana-PVA/LCbecauseof larger
amounts of crystalline junction zones, which give the gel
greater stability. Hence, the different ability of the dry s-
PVA/LC blend to trap xenon gas can be reasonably ascribed
to a more relevant amount of hydrophobic junction zones.
The
129
Xe NMR spectra of the hydrated a-PVA and s-
PVA gels and their blends showed that the signals are close
to the position of xenon in water (189ppm), and this
indicated that the swelling process leads to hydration of
all the environments probed by xenon. Contrary to the
dry samples, two resonances are observed in the
129
Xe
NMR spectrum of hydrated LC. In the pure hydrated PVA,
three resonances were observed, whereas one (a-PVA/LC)
or two (s-PVA/LC) resonances are observed in the blends
(Table 10). All of the signals are shifted downeld from
xenon in water. Furthermore, in both hydrated a-PVA and
s-PVA, the broader signals are narrower as compared to
those in the dry samples. This observation can be ascribed
to an increased mobility of the trapped xenon after the
gel swelling or to a decrease in its dipolar interactions
withwater protons. The disappearance of the signals corre-
sponding to the trapped xenon in the
129
Xe NMR spectra of
the swollen blends can be explained by strong reduction of
junction zones in the pristine matrix of the dry PVA, which
originates from the interactions between the hydrophilic
LC and water.
4.5. MRI of hydrogels and organogels
Magnetic resonance imaging (MRI) allows a lot of
possibilities, like visualization of non-homogeneities,
anisotropy effects, and surface and interface effects. In the
past years, a lot of work was done concerning the appli-
cation of MRI to swollen polymers and hydrogels [57].
MRI can be used to monitor the transport of solvent into
solid systems in real-time [416418]. It provides informa-
tion relating to the nature of the diffusion process into a
gel on a spatially resolved level with a lateral resolution
of about 10100m. An instantaneous diffusion coef-
cient can be calculated from a single image taken at a
specic time. The spatial distributions of low-molecular
weight components throughout the gel, e.g., proles of
water concentration within different crosslinked hydro-
gels [303,419], are measurable. Non-homogeneity in the
crosslink density is detectable by measuring a spatially
resolved swelling degree within a gel [319]. Summarizing,
MRI allows detecting the time and space resolved changes
in concentration of diffused species in order to measure
the network chain mobility, and the changes in chemical
composition due to transport processes.
Swelling of HPMC hydrogels was studied by MRI [175].
In the swelling hydrogel matrix tablets, the absorption of
water causes a gradual increase in the volume of the gel
and a corresponding change in the polymer concentration
whichdecreases graduallyfromtheoriginal dry-tablet face.
Quantitative knowledge of these concentration gradient
proles as functions of the swelling time allows model-
ing the swelling process. Although MRI does not detect
the HPMC polymer in these systems directly because of its
lack in molecular mobility, it is possible to determine the
polymer concentration indirectly from the concentration-
dependent effect on the T
2
relaxation times of the water
molecules.
The diffusion of water into samples of PHEMA, PTHFMA,
and copolymers of HEMA with CHMA, BMA and THFMA,
followed by formation of hydrogels, was visualized with
MRI [420422]. For PHEMA the diffusion was found to
be consistent with a Fickian model. For example, the
diffusion coefcient of water in PHEMA at 37
C deter-
mined from the proles of the diffusion front equals
1.510
11
m
2
/s (Fig. 30), which is less than the value
based upon mass uptake, 2.010
11
m
2
/s. The value
Fig. 30. NMR image of the diffusion front for water penetration into
polyHEMA hydrogel at 37
C [420]. Copyright permission fromAmerican

Chemical Society, 2001.
of diffusion coefcient for water sorption into HEMA
copolymers was found to lower by the presence of
non-polar co-monomer. For example, the diffusion coef-
cient decreased signicantly from the above-mentioned
value for PHEMA to 9.110
12
m
2
/s for poly(CHEMA-
co-HEMA), 9.710
12
m
2
/s for poly(BMA-co-HEMA) and
1.310
11
m
2
/s for poly(THFMA-co-HEMA) at composi-
tions of HEMA of 0.8. The reverse process, the water loss
fromCa-alginatehydrogel beads at acidic pH, was alsovisu-
alized with MRI [423].
The T
2
-weighted image proles of the water diffusion
front obtained from the MR images showed that a stress
was induced at the interface between the rubbery and
glassy regions which led to formation of small cracks in
this region of the glassy matrix of PHEMA and its copoly-
mers with mole fractions of HEMA greater than 0.6. Water
was capable to enter these cracks forming water pools.
For copolymers of HEMA and THFMA with mole fractions
of HEMA less than 0.6 the absence of cracks was attributed
to the ability of the THFMA sequences to undergo stress
relaxation by creep [420422].
The
1
H R
2
values in gelatin hydrogel foams measured
by the use of a multiple MRI spin-echo sequence were
found to be dependent on the echo time spacing [424].
This property, referred to as the R
2
-dispersion, originates
from the molecular self-diffusion of water within internal
eld gradients that result from the magnetic susceptibil-
ity differences between the gel and air phase. Numerical
simulations allowed studying the relation between the
foam microstructure (the mean air bubble radius) and
foam composition properties (such as magnetic suscepti-
bilities, diffusion coefcient and surface relaxation). The
R
2
-derived mean air bubble size in the hydrogel foam is
in agreement with the bubble size measured with X-ray
micro-CT. For this gel foam system the diffusion length
scale varied from1.6 to 6.3m, the dephasing length was
6.3m (for a foam system with the mean bubble radius
of 150m) and the systemlength varied between 2.1 and
3.6m(the characteristic pore dimensions).
The
1
H T
2
NMR images were observed by means of MRI
for a transverse slice of a composite PMAAc hydrogel con-
sisting of two parts with a different degree of crosslinking
[416]. The T
2
MR image was converted to the Mn
2+
ion
concentration image by using the relationship between
the Mn
2+
ion concentration and the T
2
value. From these
experiments, the spatial distribution of Mn
2+
ions in the
gel under the application of electric eld was determined
as a function of the elapsed time [417,418]. From the
migration of Mn
2+
ions, the crosslinking structure of the
interfacial region between the two parts with a different
degree of crosslinking was detected. Similarly, using such
a kind of NMR microscopy, alginate-poly-l-lysine interac-
tions within alginate hydrogels were visualized [425,426].
In the hydrogel drug delivery systems, it is impor-
tant to visualize and quantify processes of the hydrogel
formation during the standard dissolution study. Obser-
vation of dynamics of hydrogel formation, erosion and
dry core retention of controlled release, polymer-based
dosage forms are not possible using other imaging meth-
ods. MRI can be used to observe these processes in a non
destructive and non invasive way. For example, perform-
ing MR imaging simultaneously with the dissolution study
of l-dopa from the hydrodynamically balanced system in
conditions, which are the best to date simulation of in vivo
situation regarding temperature, volume, state and com-
position of dissolution media [427] allowed (i) obtaining
timeevolutionproles of cross-sectional areas of themodel
dosage form regions (i.e., diffusion, hydrogel formation,
dry core regions) fromMR images, (ii) to study together
several phenomena, which affect the controlled release,
hydrogel formationanderosion, drug dissolutionandota-
tion, and(iii) toassess andcontrast the properties of dosage
forms with the similar dissolution proles.
5. Conclusions
Determination of the structure and dynamical proper-
ties of gel systems is a complicated problem. Successful
solving of this problem utterly depends on the develop-
ment of the new effective methods of physico-chemical
analysis including the new NMR spectroscopy techniques.
The obstruction by the gel network, the hydrodynamic
interactions in the gel system, and the thermodynamic
agitation should be all considered to understand the inter-
nal mobility in gels and their morphology. Various models
and theories succeeded in describing the morphology and
internal motions under different circumstances. Enormous
progress has been made in the eld but controversies are
not uncommon. It seems also fair to say that limitations
exist for the application of the physical models and care
should be taken in the use of the models for the interpre-
tation of the obtained results. It remains difcult, if not
impossible, to estimate and predict the packing and mobil-
ity of a given component in a given system under specic
conditions. It is also important to establish a correlation
between the motions in an equilibrated state and the diffu-
sion in the real-time non-equilibrated dynamic situations,
where the swelling and the dissolution of the gel network,
the compatibility of the solvent, solute and polymer should
all be considered.
The rapid development of various NMR techniques for
thelast twodecades, especiallyintheeldof thesolid-state
and multiple-quantumNMR spectroscopy, MR microscopy
and MRI, allows investigating the more complicated gel
systems to obtain further information on the properties
of the diffusing compounds and gel networks. Studies
of this kind are capable to lead to a better understand-
ing the structure, morphology and mobility in the gel
systems.
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Progress in Polymer Science 36 (2011) 11841253

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). The tapes can further assemble,

). Subsequent aggregation (with increasing

-octoxystilbeneoate in higher alcohols,

pulseof thesecondmagnetic eldB

) of the structures are formed

pulse and the onset of the mixing period. In

C(Fig. 6b), thoughuponheating

C, the peaks became sharp [93]. This observa-

C) may be ascribed to the fact that

C (gel starts to melt), (F) 55

C (entire gel has

C. The half-height peak width ^

C by heating that is about 4

C) and then back-

=356.5J/Kmol, consistent with the implication of

C, three peaks I, II and III at 77, 71 and 65ppm for

C. The correlations of -CD protons with

obtained for trans-stilbene derivatives were

C range, and upeld shifts in the 6090

C were 93 and 78kJ/mol, while the ^S

values were determined as 240 and 197J/(mol K) [155],

=465J/(mol K), and

values for the polymer hydrogel in water are 3.4kJ/mol

values of 14.8kJ/mol and

also decreased to 4.9 and

of precipitationis reduced. The equilibrium

C (sol) and organogel at

C (gel) [182]. Copyright permission fromElsevier, 2008.

C are shown in Fig. 12 [182]. The sig-

C (sol state). Again,

C. The aromatic pro-

C indicated that H-bonds

C, implying immobilization of the

C. Upon heating, the newpeaks appeared grad-

C. At the temperature elevated up to 70

C, the signal for the pyridyl protons H

C. Notably, the posi-

C, which is consistent with the

-dithiobis(sulfosuccinimidyl propionate), the major

attached to the nitrogen of the N,N-disubstituted amide in

-tetramethylenediamine as initiators [263],

-methylenebisacrylamide. Radicals, formed after

-methylenebisacrylamide following by formation of

residing on neighboring solute molecules that are dipolar-

inverting pulses in the CPMG sequence effec-

C the shrinking of polymer segments occurs, which

C correspond only to the mobile polymer

C they reect the behav-

C. Instead, a well-resolved tri-

C are 4.5, 29 and 330ms

C, since the mobility of backbone

C, only weak NMR signal is observed (Fig. 7), indicat-

C, some backbone segments of GG gel col-

pulses spaced at half a rotor period,

pulses, in such an experiment is

pulse, denotes the duration of the magnetic eld gra-

pulse sequence is combined with

indicate that, on the one

C, and an obstruction factor is 0.325. At this

C). This was in contrast to tropoelastin hydrogel: the