Inorganica Chimica Acta 256 (1997) 69–75

13
C NMR and molecular modeling studies of alginic acid binding with
alkaline earth and lanthanide metal ions
C.M. DeRamos, A.E. Irwin, J.L. Nauss, B.E. Stout U
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA

Received 21 March 1996; revised 28 June 1996

Abstract

The binding of the binary polyuronide alginic acid to alkaline earth (Mg2q, Ca2q, Sr2q, Ba2q) and lanthanide (La3q, Pr3q, Nd3q, Eu3q,
Tb3q) ions was studied by 13C NMR spectroscopy. Divalent metal ions showed a preference for GG blocks and an increase in the extent of
binding with increasing ionic radius. Molecular modeling studies of calcium–alginate dimers support the assertion that this preference results
from the secondary structure of alginic acid. Trivalent metal ions studied showed a preference for GG blocks and an increase in the extent of
binding for both GG and MM blocks with increasing charge density. The contrast in the mode of interaction of the alkaline earth and lanthanide
metal ions with alginic acid can be associated with differences in coordination number and/or waters of hydration remaining in the inner
sphere of the lanthanide ions.
Keywords: Molecular modeling; Lanthanide complexes; Alkaline earth complexes; Alginate complexes

1. Introduction
Organic matter in soil and groundwater may play a role in
the transport of the actinides from waste repositories. Humus,
the non-living organic matter in soil, influences the retention,
migration and bioavailability of metal ions by processes such
as sorption or complexation [1]. The term humus encom-
passes a variety of substances such as polysaccharides, pro-
teins, lipids and organic acids which affect the ability of soils
to transport ions, retain water and maintain a proper pH.
Polyuronides, carboxylate-containing polysaccharide deriv-
atives, are believed to be responsible for aggregation of soil
particles, acting as a type of glue that holds soil particles
together, improving soil structure and plant growth [2,3].
The ability of polyuronides to form aggregates is related to
structural features such as length and the ability to form
hydrogen bonds through carboxylate functional groups that
enable them to bridge the spaces between soil particles [2].
Understanding how metal ions interact with polyuronideswill
help us predict the transport properties of these ions in soil
and groundwater. Due to the variety of polyuronides in
nature, we have chosen a simple model, alginic acid, for our
studies.
Alginic acid (Fig. 1) is a linear, binary copolymer of 1,4-
linked a-L-guluronic acid and b-D-mannuronic acid [4],
U
Corresponding author.
0020-1693/97/$17.00 q 1997 Elsevier Science S.A. All rights reserved
PII S0020-1693(96)05418-7
usually isolated from brown algae but is also present in some
species of bacteria [5,6]. Along the polymer chain the two
residues are arranged in an irregular blockwise pattern [7–
9]. There are three types of blocks: homopolymeric
sequences of mannuronate (MM blocks) and guluronate res-
idues (GG blocks) and a region where the two residues
alternate (MG blocks). The relative proportions of these
block types are affected by several factors [10–12] such as
the botanical source, plant maturity, collection site and sea-
sonal variations.
In solution, alginates behave like flexible coils [13]. How-
ever, upon interaction with divalent metal ions, they form an
ordered structure as evidenced by their X-ray diffraction pat-
terns [14]. Ion exchange studies [15–17] have demonstrated
the dependence of the gelling properties of alginates upon
uronic acid composition, which is referred to as the M/G
ratio. Due to the different affinities of the homopolymeric
blocks for multivalent metal ions, most of the studies of
metal–alginate interaction involve the use of fragments
enriched in the homopolymeric blocks [16,18,19]. Segments
rich in GG blocks showed enhanced binding with Ca2q ions
characterized by gel formation at a threshold number of about
20 residues [16,20], which implies a cooperative mecha-
nism. However, fractions enriched in MM blocks and alter-
nating units did not exhibit such effects. The difference in
binding among the three types of blocks is often explained in

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70 C.M. DeRamos et al. / Inorganica Chimica Acta 256 (1997) 69–75
Fig. 1. Structure of sodium alginate.
terms of alginic acid secondary structure (see Section 3).
Grant et al. [21] showed that the chain conformation of
polyguluronic acid in solution is similar to that described by
fiber diffraction studies [22], and that crosslinking involves
cooperative binding of calcium ions between aligned GG
blocks of two alginate chains [21,23,24]. In this type of
association, first referred to as the ‘egg box model’ by Grant
et al. [21], each Ca2q ion is coordinated by four guluronate
units, two from each chain. The strength and selectivity of
cooperative binding is determined by two factors: the size of
the metal ion and the ease of packing of the alginate chains
around the metal ion.
We have studied the interaction of divalent alkaline earth
metal ions and trivalent lanthanide ions with alginic acid
using 13C NMR spectroscopy. Molecular models based on
the interaction with the calcium ion are now proposed. Pre-
vious data on lanthanide luminescence studies of europium–
alginates [25,26] were used in order to help model the inter-
action of trivalent lanthanides with alginic acid.
2. Experimental
2.1. Materials
Alginic acid isolated from Macrocystis pyrifera was pur-
chased from Sigma and used without further purification.
Reagent grade alkaline earth metal chlorides were obtained
from Fisher (CaCl2 and BaCl2) and Baker (SrCl2). Lan-
thanide oxides (99.9%) were obtained from Sigma (La2O3),
Aldrich (Eu2O3, Pr6O11), Matheson, Coleman and Bell
(Nd2O3) and Alfa Products (Tb4O7). All other reagents used
were analytical grade.
2.2. Preparation of metal ion stock solutions
2.2.1. Alkaline earths
1 M stock solutions of alkaline earth metal chlorides were
prepared by dissolving the chloride salt in D2O (99.9% D)
and standardized either by direct EDTA titration (for CaCl2)
or back titration of the metal–EDTA complex with standard
Mg(ClO4)2 using Eriochrome Black T indicator. The solu-
tions were buffered to pH 10 with NH3/NH4Cl and the deter-
minations were done in triplicate.
2.2.2. Lanthanides
1 M stock solutions of lanthanide perchlorates were pre-
pared in D2O following the method described elsewhere[27]
for the preparation of aqueous lanthanide salts. For the prep-
aration of terbium perchlorate, the mixture was refluxed for
6 to 8 h after the first addition of conc. HClO4 to ensure
complete dissolution of the oxide prior to evaporation of the
excess acid. The solutions were standardized by EDTA titra-
tion using xylenol orange indicator and hexamethylene tetra-
mine buffer [28].
Caution: Ionic perchlorates such as alkaline earth and
lanthanide perchlorate salts are not explosive in general.
However, exposure of these pure compounds to easily oxi-
dized organic matter at high temperature greatly increases the
risk of fire or explosion [29].
2.3. Preparation of metal alginate samples
Metal–alginate samples were prepared by first suspending
750 mg alginic acid in D2O then neutralizing 75% of the
carboxylic acid (calculated from the carboxylic acid capac-
ity) by the addition of 1.0 M NaOD. Because polymer solu-
tions require longer equilibration times, the resulting solution
was stirred for 30 min and then heated to 708C prior to the
addition of metal ion. An appropriate amount of preheated
0.050 M alkaline earth metal or 0.015 M lanthanide metal
solution was slowly added to achieve the desired metal load-
ing (mequiv. metal ion/mequiv. COOH=100%). D2O was
again added to a final volume of 3.00 ml. For all metals
studied, except terbium, six samples were prepared with 4, 8,
12, 16, 20 and 24% metal loadings. Due to extensive broad-
ening of peaks at high metal loadings, Tb3q metal loadings
were 4, 6, 8, 10 and 12%. All samples were equilibrated for
19 to 24 h in a sand bath at a temperature of 50 to 608C prior
to NMR acquisitions. The final pH of the solutions measured
at room temperature ranged from 3.8 to 4.2.
2.4. NMR spectroscopy
All 1H and proton-decoupled 13C NMR spectra were
acquired on a Bruker AMX 400 NMR spectrometeroperating
at 400.16 and 100.62 MHz, respectively. 1H spectra were
acquired using 32 K data points, a spectral width of 4032 Hz,
a pulse repetition time of 2.5 s and a total of 8 scans were
collected. 13C spectra were recorded using 32 K data points,
a spectral width of 31 250 Hz, a 908 pulse and a total of 1600
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scans. In order to quantitatively compare signal intensities of
the different carbons, a long pulse repetition time of 10.5 s
[30] was used to ensure complete relaxation of the slow-
relaxing carboxylate (C6) carbons. A probe temperature of
708C was used in order to decrease peak broadening, evident
at temperatures lower than 708C in the 1H NMR spectra of
sodium alginate, due to viscosity [30]. All determinations
were performed in duplicate.
Peak intensities reported as average G’s and M’s were
averages of the most resolved C1 and C2 carbons of GG
blocks and C1, C2 and C4 carbons of MM blocks, where G/
M1 is the anomeric carbon and G/M6 is the carboxylate
carbon (see Fig. 2). Reported errors were the standard devi-
ation between at least two determinations.
2.5. Characterization of alginic acid
2.5.1. Carboxylic acid capacity
The carboxylic acid capacity of alginic acid was deter-
mined by acid–base titration. A known excess of 0.050 M
NaOH was added to a suspension of 75 mg of alginic acid in
water, the mixture stirred until all the alginate was dissolved,
and the unreacted base was back-titrated with 0.020 M HClO4
using phenolphthalein as indicator. The determination was
done in triplicate.
2.5.2. Uronic acid composition and block length
A sample containing 75 mg alginic acid was fully neutral-
ized by titration with 1.0 M NaOD. D2O was added to achieve
a final concentration of 25 mg alginic acid/ml D2O. The
sample was equilibrated for at least 1 h prior to NMR acqui-
sition. The uronic acid composition was determined by 13C
NMR spectroscopy as described above, except that the pulse
repetition time was decreased to 2.5 s and a total of 16 000
scans was collected. From the areas of the eight triad fre-
quencies arising from the anomeric carbons, the relative
amounts of guluronate and mannuronate residues and the
average length of each block type were obtained following
Fig. 2. 13C NMR spectrum of alginic acid.
71
the calculations of Grasdalen et al. [31]. The term triad refers
to three consecutive uronic acid residues in an alginate chain.
The reported composition and block lengths were the average
of two determinations.
2.5.3. Average molecular weight
The average molecular weight was determined from vis-
cosity measurements using a Cannon-Fenske capillary vis-
cometer on a thermostated water bath kept at 25.0"0.18C. A
200 ml stock solution containing 0.10 g alginate/ml water
was prepared by first adding 1.0 M NaOH to a suspension of
alginic acid in water to neutralize 100% of the carboxylate
groups. NaCl was then added to achieve a total concentration
of 0.1 M NaCl. From the stock solution, eight different con-
centrations of sodium alginate were prepared ranging from
0.02 to 0.09 g/ml. In all these dilutions, 0.1 M NaCl was used
as solvent. All viscosity measurements were performed in
triplicate. The reported viscosity-average molecular weight
was the result of two determinations.
Solution viscosity is empirically related to molecular
weight for linear polymers according to the Mark-Houwink-
Sakurada equation [32–34]:
[h]sKMva (1)
where [h] is the intrinsic viscosity in dL/g and Mv is the
viscosity-average molecular weight in daltons. The constants
a and K depend on the solvent and polymer type.
Eq. (1), with values of Ks7.3=10y5 and as0.92 as
determined by Martinsen et al. [35], was used to calculate
the viscosity-average molecular weight of our sample.
2.6. Molecular modeling
Molecular modeling was accomplished using the software
packages InsightII and Discover, both from Biosym Tech-
nologies. The AMBER forcefield [36] with the extensions
for polysaccharides developed by Homans [37] was applied.
Model dimers of mannuronic acid and guluronic acid were
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subjected to steepest descents energy minimization in vacuo
with a distance-related dielectric constant until the minimum
energy was reached. Pairs of the resulting dimers were placed
in different orientations with respect to the chain direction
about the calcium ion: parallel, antiparallel and perpendicu-
lar, with the antiparallel having the lowest binding energy.
Each dimer pair was minimized again using the steepest
descent method.
interaction of heteropolymeric MG blocks with metal ions
will be the subject of future publications.
Two interrelated aspects of binding may be discussed:pref-
erence of a metal ion for a particular block type and the extent
or strength of the metal–alginate interaction. The extent of
binding is roughly analogous to the stability constant for

3. Results and discussion

3.1. Characterization of alginic acid

A carboxylic acid capacity of 4.50"0.03 mequiv./g was

obtained for alginic acid. This translates into an equivalent
weight of 222 g/equiv. which is higher than the theoretical
weight of 176 g/equiv. for a hexuronoglycan [38]. However,
commercial alginates normally have high equivalent weights
due to water that is strongly absorbed even if samples have
been rigorously dried.
Using Eq. (1) and the constants determined by Martinsen
et al. [35] for Macrocystis pyrifera we obtained a viscosity-
average molecular weight of ;16 700"900 Da, which Fig. 3. 13C NMR spectra of calcium–alginate. Metal loadings of: (A) 4, (B)
12 and (C) 20%, respectively.
implies an average chain length of 75 residues. (Note: pre-
viously [25] we reported a viscosity-average molecular
weight of 7000 Da for our sample using the constants from
the work of Donnan and Rose [39], which were later deter-
mined by several authors to be different for different species
of alginates [35,40].) The determined alginate composition
of 45"3% guluronate and 55"3% mannuronate residues
was very close to the composition reported by Grasdalen[41]
using the same method for alginate isolated from M. pyrifera.
Both the GG and MM blocks gave the same average block
length consisting of about five residues.

3.2. Factors influencing metal–alginate complexation

The formation of gel upon the addition of a polyvalent

metal ion to a solution of alginic acid results in a significant
decrease in the mobility of alginate residues that are in the
gel phase. This restriction of molecular motion decreases the
nuclear relaxation time to a great extent [42] giving rise to
broad 13C NMR signals. Because of this extensive broaden-
ing, the spectra show only the signals arising from the
unbound or free alginate residues.
As metal ion loading increased from 4 to 24%, the peak
intensities decreased, indicating that more of the alginate
residues are binding to the metal ion. This can be seen in the
spectra for calcium alginate (Fig. 3). For all the metal ions
studied, the change in peak intensity was measured, normal-
ized to the lowest metal ion loading in order to compare data
obtained with different metal ions, and plotted againstpercent
metal ion loading (Fig. 4). Only the binding of the homo- Fig. 4. Percent metal ion loading vs. normalized peak intensity for (a)
polymeric GG and MM blocks is discussed in this paper. The calcium–alginate and (b) europium–alginate.

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 C.M. DeRamos et al. / Inorganica Chimica Acta 256 (1997) 69–75 73

metal–alginate complexation while the preference may be a total of six binding sites (Fig. 5) while binding with MM
thought of as the ratio of the stability constants for metal ion blocks involves a ring oxygen (O5) in each dimer instead of
binding with GG blocks and MM blocks. Therefore, the pref- the hydroxyl oxygen (O2) found with GG dimers (Fig. 6).
erence of metal ion for GG or MM blocks is a consequence Because hydroxyl oxygens are stronger Brønsted-Lowry
of the extent of its binding to a particular homopolymeric bases than ether oxygens, a stronger interaction between
block. metal ions and GG blocks is expected. These molecular mod-
The preference of a metal ion for a particular block type els further support the observed positive GG-preference of
may be determined from plots of normalized peak intensity alkaline earth metal ions.
versus metal loading, measured for both MM and GG blocks. In the case of Ca2q, the extent of binding for GG blocks
We refer to the gap between the curves of the MM and GG increases with increasing Ca2q loading. For MM blocks,
data as the GG-preference. When there is more binding to there is significant binding only at high metal loadings at
GG than MM blocks, the GG-preference is positive. For which point the GG binding sites may be saturated. The more
instance, both Ca2q and Eu3q showed positive GG prefer-
ence (Fig. 4), but the gap between the two curves differ as Table 1
Selected bond distances (Å) for Ca2q–GG and Ca2q–MM dimers
will be dicussed (vide infra).
The extent of binding may be determined from the shape Interaction GG dimer distance Interaction MM dimer distance
of the curves of peak intensity versus metal ion loading. Since
the measured peak intensities represent the amount of Ca–O6 2.20 Ca–O69 2.19
unbound alginic acid residues only, a negative slope indicates Ca–O29 2.29 Ca–O3 2.24
Ca–O39 2.40 Ca–O59 2.41
increasing extent of binding with increasing amount of metal Ca–O 2.96 Ca–O 3.45
ion added. The extent of binding for polyelectrolytes is Ca–O59 4.73 Ca–O29 4.34
dependent in part on metal ion concentration. For instance, Ca–O3 6.45 Ca–O2 4.75
as the repulsive forces of the negatively charged carboxylate
group of each uronic acid residue are neutralized, the alginate
structure becomes more condensed [43]. These conforma-
tional changes may influence the extent of metal ion binding
if it is dependent on the geometry of the binding site. Also,
once the binding sites of one type of homopolymeric block
are filled, binding to the other block may begin to occur.
3.3. Alkaline earth ions

Our previous studies have shown that magnesium does not

form inner-sphere complexes with alginates, even at high
(53%) metal loadings [25]. Magnesium, with rs0.72 Å for
the hexa-coordinate ion [44], may not be able to effectively
crosslink alginate chains. From the plot of normalized peak Fig. 5. Molecular model of two GG dimers with Ca2q. The six close inter-
intensity for Ca2q–alginate (Fig. 4(a)) it is apparent that actions of calcium ion with three oxygens from each dimer are indicated
Ca2q exhibits a positive GG-preference. A positive GG-pref- with solid lines, (see Table 1 for interaction distances).
erence was found for Sr2q and Ba2q as well. The GG-pref-
erence of alkaline earth metal ions appears to depend on the
alginic acid secondary structure. The diaxial linkage pattern
of guluronate residues results in a buckled structure [22]
with deep cavities for the metal ions to occupy while man-
nuronate residues are linked diequatorially, resulting in a flat
ribbon-like structure [45] with shallow cavities. Further-
more, the type of oxygen atoms involved in binding with the
metal ion may affect the binding preference.
Guluronate and mannuronate dimers were used to model
the interaction of Ca2q with GG and MM blocks (Figs. 5 and
6, respectively). A total of six close interactions (F2.41 Å)
between Ca2q and the alginate oxygens for each block type
was obtained (Table 1). The two block types differ only in
the type of oxygens that are in close proximity to the metal
ion. For GG blocks, a carboxylate oxygen (O6) and two Fig. 6. Molecular model of two MM dimers with Ca2q, (see Table 1 for
hydroxyl oxygens (O2 and O3) in each dimer are involved, interaction distances).

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74 C.M. DeRamos et al. / Inorganica Chimica Acta 256 (1997) 69–75
Fig. 7. Metal ion charge density vs. normalized peak intensity for GG and
MM blocks at 8% metal loading.
extensive binding of GG blocks as compared to MM blocks
was consistent with the work of Kohn and Larsen [16]. Using
activity coefficient as a measure of the interaction between
calcium ion and alginate fractions enriched in GG and MM
blocks, they concluded that the linear charge density of the
polyion, among other factors, influences the strength of bind-
ing between the metal and the polyion. The smaller distance
between negatively-charged carboxylate groups in the
buckle-shaped guluronate sequences results in a higher
charge density and a stronger interaction with the positively-
charged counterion, thereby decreasing the activity coeffi-
cient of calcium ion in solution. In line with this argument,
less extensive binding with calcium ion is expected for MM
blocks. Because of the flatter structure of mannuronate
sequences, the unit charges are farther apart, lowering the
MM block charge density as compared to that of GG blocks.
There is a significant difference in the extent of binding of
Ca2q, Sr2q and Ba2q for GG blocks at low metal loadings
(Fig. 7). The extent of binding at low metal loadings with
GG blocks increases with increasing ionic radius and follows
the order Ba2q)Sr2q)Ca2q4Mg2q. This result is con-
sistent with that obtained by Haug and Smidsrod [18] from
equilibrium dialysis experiments in which competition
between divalent metal ions was studied. Shorter and there-
fore stronger ionic bonds between the alginate oxygens and
the larger metal ions would be expected. At high metal load-
ings (e.g., 24%), there is no difference in the binding of the
three divalent ions, probably because maximum binding is
achieved. This could be due to a saturation of the alginate
binding sites or conformational changes in the alginate
structure.
3.4. Lanthanide ions
All the trivalent lanthanides studied exhibited a less posi-
tive GG-preference than that of the alkaline earth metals. This
is evident when the binding of Ca2q and Eu3q, for instance,
(Fig. 4), are compared. These differences are not due to a
decrease in GG block binding by the lanthanides but rather
an increased participation by MM blocks. The positive GG-
preference has been explained in terms of alginic acid sec-
ondary structure (vide supra). The fact that there is
significant binding of the lanthanides and virtually no binding
of the alkaline earths to MM blocks indicates that the mode
of binding for these groups of metal ions differs. We attribute
this to additional waters of hydration in the inner sphere of
the lanthanide ions.
Our earlier studies of Eu3q–alginate complexes by lan-
thanide luminescence spectroscopy (LLS) confirmed that
crosslinking of the alginate chains, and therefore gelation,
appears to require inner sphere metal–ligand interactions
[25,26]. The Eu3q excited states can be quenched by
vibronic coupling with the OH oscillators of water or
hydroxyl groups in the inner sphere of the europium ion.
Furthermore, these oscillators quench independently, making
it possible to quantify the remaining inner-sphere oscillators
by comparison with model compounds [46]. Complexation
with alginate necessitates at least a partial dehydration of the
europium aquoion, Eu(H2O)9 3q. LLS measurements show
that after complexation only six OH oscillators remain in the
inner sphere of the europium ion, consistent with a combi-
nation of uronic acid hydroxyl groups (one OH oscillator
each) and water molecules (two OH oscillators each). The
carboxylate groups do not effectively quench the Eu3q
excited states. However, based on the model for Ca2q inter-
action with GG and MM blocks, two carboxylate groups must
be coordinated to the lanthanide ion as well. Therefore,
assuming that the coordination number is constant from La3q
to Tb3q, a lanthanide ion with one or two inner sphere water
molecules may not pack as easily into the GG and MM block
cavities compared to a completely dehydrated alkaline earth
ion. Binding may be more dependent on the cation charge
density than on the geometry of the alginate chain.
In contrast to the divalent ions, as ionic radius decreases
across the lanthanide series from La3q to Tb3q, metal–algin-
ate binding increases for both GG and MM blocks, reaching
a maximum with Tb3q (Fig. 7). The size of a trivalent ion
seems to be a less important contributing factor to alginate
complexation than cation charge density. Perhaps this is
partly due to the fact that the variation in ionic radius from
La3q–Tb3q (1.22–1.04 Å) is about 17%, which is about half
that of the alkaline earths studied Ca2q–Ba2q (1.00–1.35
Å). Analogous plots at higher metal loadings show the same
trend, indicating that conformational changes in alginate
structure play little or no role in lanthanide ion binding.
4. Conclusions
Alkaline earth and lanthanide ion complexation of algin-
ates are dependent on different factors. The divalent metal
ions show a binding preference for GG block residues over
MM block residues. This preference may be explained in
terms of the alginic acid secondary structure and is also sup-
ported by the molecular models. The extent of binding with
GG blocks increases with increasing ionic radius at low metal
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loadings, while at high metal loadings it appears to have
reached a maximum for all three metals. Small but significant
binding with MM blocks is observed only at high metal
loadings.
For the trivalent lanthanide ions, there is again a preference
for GG blocks over MM blocks. However, a different inter-
action between the metal ion and the alginate chain is occur-
ring as indicated by the relatively strong binding to MM
blocks. Within the range of metal ion loadings studied, the
extent of binding with both GG and MM blocks increases
with increasing charge density of the metal ion, reaching a
maximum with Tb3q. This contrast between the alkalineearth
and the lanthanide metal ions may be due to differences in
coordination number, and the fact that one or two waters of
hydration remain in the inner sphere of the lanthanide ion
after complexation with alginate.
Acknowledgements
This project was funded in part by the National Science
Foundation (No. CHE-9109396) and the Petroleum
Research Fund ( No. 24372-G3). The authors wish to thank
Dean Peterman for the LLS measurements.
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Journal: ICA (Inorganica Chimica Acta) Article: 5418