Solid State Nuclear Magnetic Resonance 15 Ž2000. 239–248
www.elsevier.nlrlocatersolmag
Short communication
13
C CPMAS studies of plant cell wall materials and model
systems using proton relaxation-induced spectral editing
techniques
Hui-Ru Tang ) , Yu-Lan Wang, Peter S. Belton
Institute of Food Research, Norwich Research Park, Colney Lane, Colney, Norwich NR4 7UA, UK
Received 20 October 1999; accepted 6 December 1999
Abstract
The solid state 13 C CPMAS NMR spectra of plant cell walls are often complex owing to superposition of resonances
from different polysaccharides and the heterogeneity of the cell wall assembly. In this paper, we describe the application of a
set of proton relaxation-induced spectral editing ŽPRISE. experiments which combine 1 H relaxation properties ŽT1, T1r , T2 .
with 13C high resolution spectroscopy ŽCPMAS. to relate the dynamics of the plant cell walls and model systems to their
domain structural details. With PRISE it has been found that in plant cell wall materials, cellulose is always associated with
the long components of spin–lattice relaxation in both the laboratory and rotating frames whereas non-cellulose polysaccharides Žpectin and hemicellulose. are associated with the short ones. For the proton T2 relaxation, cellulose is only associated
with the short component Žbelow 20 ms., pectin contributes to both the short component and the long one. q 2000 Elsevier
Science B.V. All rights reserved.
Keywords:
13
C CPMAS; Plant cell walls; Proton relaxation; Cellulose; Pectin; PRISE
1. Introduction
The structural heterogeneity of plant cell walls is
of great importance for their functions both in living
cells and the post-growth utilisation of plant materials. Typically, the length scale of the heterogeneity is
such that proton spin diffusion is inefficient, which
often results in multiple-components for some of the
proton NMR relaxation times w1–8x ŽT1 , T1r , T1D ,
)
Corresponding author. Tel.: q44-1603-255000; fax: q441603-507723.
T2 .. Consequently, proton NMR provides a good
technique to probe the properties of the heterogeneity of the cell walls non-invasively w1,2x. An approximate theory w9,10x, which relates the spin diffusion
properties to domain size and geometry, may be
employed together with experiments such as the
Goldman–Shen experiment w11x. However, proton
NMR alone cannot give an unambiguous account of
the structural characteristics since it does not readily
distinguish chemical entities in the solid state. For
this purpose, 13 C CPMAS spectroscopy offers a much
higher chemical resolution for cell walls w12–20x but
is intrinsically less useful in studying heterogeneity.
0926-2040r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 6 - 2 0 4 0 Ž 9 9 . 0 0 0 6 4 - 8
240
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
In the heterogeneous systems such as cell walls,
neither proton relaxation time measurements nor 13 C
high resolution spectroscopy alone can provide information on both the chemical structure and dynamic
properties of the different domains.
Proton relaxation times are critically important in
13
C CPMAS experiments since CPMAS experiments
involve magnetisation transfer from protons to 13 C
nuclei w21x. If multiple components are present in the
proton relaxation processes, proton magnetisation can
be prepared in such a way that the magnetisation of
protons having different relaxation times is separated
to some extent before being transferred to 13 C.
Therefore, the combination of both techniques, at
least in theory, ought to provide more potent information about dynamics and related chemical structure in one set of experiments. Based on this theory,
Zumbulyadis w22x described a ‘‘selective carbon excitation’’ method to detect spatial heterogeneity using proton T1 , T1r and T2 . Similarly, this strategy
was used to measure proton relaxation times via 13 C
CPMAS w23x or to estimate domain sizes w24x. The
usefulness of the ‘‘delayed contact’’ method w23,25x
has been clearly demonstrated in studies of synthetic
polymers w25x and in measuring cellulose crystallinity w26,27x. In this experiment, components with
different proton T1r can be separated in a 13 C CPMAS experiment.
As a part of a systematic investigation of plant
cell wall materials, we demonstrated the power of
both the proton relaxation time measurements w5–
7,28x and 13 C MAS spectroscopy w20x in probing
molecular structure and dynamics of plant cell walls
and model systems w5–7,20,28x. It was concluded
that while cellulose retained its structural rigidity
even when the water content was as high as 410%
Žwt.rwt.., the mobility of pectic materials was much
more dependent upon the water content w5,7,20x.
Even when water content was as low as 20%, a
substantial amount of pectin was mobilised w5,7,20x.
What remains to be explained is the mechanism of
the coupling of cellulose rigidity and pectin mobility
in the real plant cell walls, in which water is one of
the most important components. However, evidence
obtained so far w5,20,29,30x appears to support a
model in which the rigidity of cellulose provides
strength and shape to the cells whereas the mobility
of non-cellulose polysaccharides is important for
other properties such as porosity, mass transportation
and signalling w31x.
In our previous study, we found that nearly all
wet Ž20–410% water. cell wall materials of both
potatoes ŽPB. w5,6x and Chinese water chestnuts
ŽCWC. w6,7x showed multiple-component relaxation
processes. In this paper, we report some results on
relating the proton relaxation times of plant cell
walls and model systems with their structural characteristics by means of combining 1 H relaxation measurements with 13 C high resolution spectroscopy in
the solid state. The principles of our experimental
techniques are similar to that of Zumbulyadis w22x.
However, we choose to adopt the version of pulse
sequence for T1r-based spectral editing, first described by Sullivan and Maciel w23x and later named
the ‘‘delay contact’’ experiment w25,27x. Furthermore, we propose that the collection of methods
described be collectively referred to as: PRISE Žproton relaxation-induced spectral editing..
2. Experimental
2.1. Materials
A mixture Ž1:1, wt.rwt.. of cellulose and pectin
was prepared by mixing a-cellulose ŽSigma. and
pectin ŽSigma p9561, degree of methylation 95%..
This sample was called CP11. a-Cellulose was suspended in a quantity of distilled water for 2 h
followed by stirring for a further two. A similarly
prepared pectin suspension was added to the cellulose suspension with stirring and the mixture was
further stirred with a magnetic stirrer for 4 h. After
lyophilisation, the mixture was re-suspended in water
and stirred for 4 h and freeze-dried again. This
mixture was freeze-dried from D 2 O three times to
replace exchangeable protons. Plant cell wall materials of PB and CWC were prepared and exchanged
with D 2 O as described previously w5,20x. Hydration
was carried out in vapour over D 2 O in a sealed jar.
D 2 O exchange and hydration was employed in order
to avoid complications of exchangeable protons and
protons from water. Water content expressed here is
defined on wt.rwt. basis, i.e., as grams water per
100 g of solid materials.
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
241
2.2. NMR measurements
All NMR measurements were carried out on a
Bruker MSL-300 spectrometer with a double bearing
magic angle spinning probe head. Proton 908 pulse
length was 4 ms and sample spinning rate was
3.5–4.5 kHz. Since sample spinning can affect proton relaxation times w32–34x, we ensured that all
samples were spun at the ‘‘magic angle’’ and relaxation time measurements and 13 C MAS spectra were
conducted at the same spinning rate.
Proton relaxation times, T1 , T1r , T1D and T2 , were
measured through decoupling channel using standard
inversion recovery, spin-locking, Jeener–Broekaert
w35x and solid echo w36x pulse sequences, respectively. For T1 measurements, the relaxation delay
was chosen as described previously w5,37x so as to
make multiple relaxation processes easier to be detected. Relaxation times were extracted by curve-fitting with a software TableCurvee ŽJandel Scientific.
on a Pentium PC.
13
C CPMAS spectroscopy was conducted with a
single contact time of 1.2 ms and the PRISE experiments were carried out as described in the Fig. 1. For
proton T1 partially relaxed experiment, the relaxation delay, t , was chosen such that the short component was nullified while the long component remained inverted. Two other experiments were carried out to verify this experiment by choosing t
equal to tnull of the long component Žthe short
component was substantially relaxed. or between
tnull of the short component and the long component
Žthe short T1 component was partially relaxed and
recovered whereas the long component remained
inverted.. For T1r and T2 partially relaxed experiments, we chose a relaxation delay such that the
short T1r and T2 components decayed below 1% of
its initial intensity. Although this approach is not
necessarily the best Žas it often leads to poor
signalrnoise ratio., this method can avoid any artificial bias arising from spectral-subtraction.
3. Results and discussion
3.1. Pulse sequences for PRISE
Fig. 1 illustrates the pulse sequences of the PRISE
experiments in which Ža. shows the way to combine
Fig. 1. Pulse sequences for the proton relaxation-induced spectral
editing ŽPRISE.. Ža. Proton T1 selective when t is tnull of one of
T1 components; Žb. proton T1r selective when t is chosen to let
the magnetisation of one of the short T1r component decay
substantially so that the long one becomes the major signal
contributor Žsee experimental section for details.; Žc. proton T2
selective when t is chosen to let the short T2 component decay
substantially so that the long one becomes the major signal
contributor Žsee experimental section for details.; Žd. proton T1D
selective when t is chosen to let the short T1D component decay
substantially so that the long one becomes the major signal
contributor Žsee experimental section for details..
a proton inversion-recovery sequence with a CP
sequence, enabling multiple-components of proton
T1 to be differentiated. The approach is the same as
described by Zumbulyadis w22x, where, after inversion, an appropriate recovery delay is chosen such
that the magnetisation of one of T1 components is
zero whilst the remainder remains inverted or partially recovered. Fig. 1b shows the combination of a
standard proton spin-locking w38x and CP sequences
to separate subspectra of domains having different
T1r w23x. In contrast, Fig. 1c shows the combination
of the proton T2 relaxation with CP so that the
components of proton T2 relaxation can be differen-
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
242
tiated w22,24x. This sequence can be regarded as the
one-dimensional version of the WISE experiment
w39x. Fig. 3d shows that proton magnetisation can be
prepared using Jeener–Broekaert sequence w35x to
enable two components with different spin–lattice
relaxation times in the local dipolar field, T1D , to be
separated before cross-polarisation.
Although the multiple-component processes for
T1D have been observed for cell wall materials before w1–4,8x, exploration of T1D in the same way is
limited since T1D is dependent on the sample spinning rate w40–43x ŽTable 1.. Sample rotation always
leads to a drastic speeding up of dipolar relaxation
when the sample spinning rate, vr Ž; 4 kHz in our
case., is much smaller than the local dipolar field
frequency Ž; 40 kHz., v L . This is because rotation
in the magnetic field causes a mixing of the dipolar
levels. For a rotating sample, the total observed
y1 .
w
x
relaxation rate, ŽT1D
obs , can be expressed as 40,43 :
y1
y1
y1
Ž T1D
. obs s Ž T1D
. st q Ž T1D
. rot
Ž 1.
where
y1
Ž T1D
. rot s
3 vr2
Ž vr2 < v L2 . .
vL
Ž 2.
y1 .
For the relaxation rate for a static sample, ŽT1D
st ,
in the order of 3 ms, and vr in 1–4 kHz, the
relaxation contribution from the sample rotation,
y1 .
ŽT1D
rot , can be up to an order of magnitude greater
y1 .
than ŽT1D
st . Such a dominance of the rotational
contribution to the observed T1D relaxation makes it
impossible to apply T1D in a fashion similar to that
in the other relaxation processes. When vr ) v L , the
approach described in Eq. Ž2. is not valid and the
Table 1
T1D data for a-cellulose and a-celluloserpectin mixture
vr ŽkHz.
T1D Žms.
CP11 Ždry.
0
a-Cellulose Ždry.
0.7
1.5
4.1
0
1.0
2.0
4.0
0.093"0.025 Ž18%. a
2.52"0.16 Ž82%.
0.439"0.01
0.137"0.01
0.037"0.002
2.90
0.32
0.087
0.032
a
Percentage proportion of the components.
relaxation of the average dipolar energy would be
equal to that of the static sample again w43x. However, such sample rotational rates Že.g., 100 kHz. are
not available at present and even if they were, crosspolarisation would become very different because of
the reduction in proton second moments. Nevertheless, in some cases where chemical shifts of the
resonances from two T1D components differ significantly, T1D -based PRISE experiments could still be
of some value even without sample rotation.
The PRISE experiments can be carried out to
measure the proton relaxation times via 13 C detection
with a CPMAS experiment w23,24,29,44x, giving useful information from the resolved carbons. However,
this indirect measurement often suffers from the
overlapping of the carbon resonances, especially in
the case of mixed biopolymer systems such as plant
cell walls. In addition, poorer sensitivity of carbons
compared with protons w44x, often requires a much
longer experimental duration to achieve reasonable
signalrnoise ratio for 13 C spectra in order to measure proton relaxation times reliably. Therefore, great
care has to be taken to avoid errors due to poor
signalrnoise ratio in 13 C experiments. It is apparent
that the PRISE experiments can be most conveniently implemented to separate carbon resonances
of the chemical entities having different proton relaxation times if the values of the proton relaxation
times are available.
For reasons discussed above, we have deliberately
chosen to measure these relaxation times directly
from proton signals and relaxation data are tabulated
in Table 2. Both cellulose and pectin alone showed
single T1 , but two components for T1r and T2 ŽTable
2.. For cellulose, the percentage of the long T2
component roughly agrees with that of water protons
in the total proton pool. For pectin, the percentage of
the long T2 component is clearly more than just that
of water, which is about 20% of the total protons.
This implies that some of the pectin protons are in
the long component as well. However, the percentage of components for T1 and T1r cannot be simply
accounted for in the similar manner due to mixing
resulting from spin diffusion.
3.2. Mixtures of cellulose and pectin
Fig. 2 shows the spectra from application of the
PRISE techniques to a model system, CP11, consist-
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
243
Table 2
Proton relaxation data for plant cell walls and model systemsa
T1 Žs.
a-Cellulose Ž8% H 2 O.
Pectin ŽDM s 95%; 11% H 2 O.
CP11 Ždry c .
CP11 Ž65% D 2 O, wt.rwt..
CWC Ždry.
CWC Ž88% D 2 O, wt.rwt..
PCW Ždry.
PCW Ž116% D 2 O, wt.rwt..
1.063
0.995
0.423 Ž37%.; 0.147 Ž73%.
0.757 Ž60%.; 0.236 Ž40%.
0.134 Ž75%.; 0.022 Ž25%.
0.171 Ž95%.; 0.005 Ž5%.
0.787Ž79%.; 0.219Ž21%.
0.676 Ž97%.; 0.140 Ž3%.
T2 Žms.
T1r Žms.
b.
13.22 Ž84% ; 3.58 Ž16%.
7.88 Ž83%.; 1.80 Ž17%.
10.48 Ž73%.; 2.39 Ž27%.
24.78 Ž76%.; 3.95 Ž24%.
9.81 Ž69%.; 2.785Ž31%.
22.21 Ž63%.; 3.69 Ž37%.
7.95Ž73%.; 2.59Ž27%.
24.16 Ž56%.; 4.35 Ž44%.
17.3 Ž90%.; 281 Ž10%.
18.9 Ž72%.; 249 Ž28%.
17.7 Ž91%.; 125.0 Ž9%.
19.5 Ž85%.; 130.0 Ž15%.
16.5 Ž92%.; 171 Ž8%.
19.5 Ž63%.; 103 Ž37%.
16.5Ž92%.; 147Ž8%.
20 Ž49%.; 236 Ž51%.
a
Measured with sample spinning at 4 kHz at the magic angle, for details please see experimental section.
Proportion of the components in percentage.
c
Dry sample contains less than 5% water.
b
ing of a-cellulose and pectin Ž1:1 wt.rwt... Proton
relaxation time measurements showed that CP11 had
two components for the proton T1 and T1r relaxation
processes, but effectively only one for T2 ŽTable 2..
The CPMAS spectrum of dry CP11 ŽFig. 2a. showed
signals from both pectin and cellulose as indicated.
Fig. 2. PRISE spectra of a dry model system CP11 Žsee experimental section for details.. Ža. Standard CPMAS spectrum; Žb.
proton T1 partially relaxed using sequence in Fig. 1a, t s 0.102 s,
phase-shifted to make cellulose peaks face upwards; Žc. proton T1r
partially relaxed spectrum using sequence in Fig. 1b, t s13 ms.
Using spectral editing techniques, PRISE, we were
able to separate those components according to their
proton relaxation times. Fig. 2b shows the proton T1
partially relaxed 13 C CPMAS spectrum Žsee figure
legend for details. in which the magnetisation of the
short T1 component was allowed to recover to slightly
above zero before cross-polarisation. Since this spectrum was phase shifted 1808 to show the intense
resonances as peaks, it was apparent that all cellulose
signals remained inverted whilst the pectin signals
were slightly recovered. Therefore, the long 1 H T1
component is associated with cellulose including both
the crystalline Ž89 ppm. and para-crystalline or surface form Ž83 ppm. w26,27x. Pectin, whose presence
is indicated by carbonyl peaks Ž170–175 ppm. and a
methoxyl peak Ž53 ppm., gives rise to a very small
inverted signal and is thus associated with the short
1
H T1 component ŽFig. 2b.. Fig. 2c shows that the
long 1 H T1r component is associated with crystalline
cellulose. However, there were some signals from
paracrystalline or surface cellulose present, but the
intensity of these is less than those in the 1 H T1
partially relaxed subspectrum ŽFig. 2b.. This implies
that spin diffusion between protons in the cellulose
crystalline microfibrils and those on the surface is
efficient in the time scale of T1 but less efficient in
the time scale of T1r . This is consistent with the fact
that cellulose microfibrils have a diameter of up to
40 nm w2x. In addition, lack of a strong pectin signal
intensity in the subspectra of the long 1 H T1 and T1r
components suggests that the interactions between
cellulose and pectin are such that spin diffusion is
inefficient.
244
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
In the transverse relaxation of this mixture, most
of the protons Ž91%. had a fast decay. The slow T2
consists of both incompletely removed water protons
and probably signal from pectin as in the case of
pectin alone. The short T2 component should include
contributions of cellulose and the most of pectin
since the protons from pectin account for about half
of the total proton population. However, T2 partially
relaxed subspectra Ždata not shown. could not assist
clear assignment for the long component of T2 due
to poor signalrnoise ratio resulting from its small
population Ž9%..
Fig. 3 shows 13 C PRISE spectra of CP11 containing 65% Žwt.rwt.. D 2 O. A number of observations
can be made from these spectra. First, although some
of the pectin signals became less intense compared
with the dry sample ŽFig. 2a., due to hydration-in-
Fig. 3. PRISE spectra of CP11 containing 65% D 2 O. Ža. Standard
CPMAS spectrum; Žb. proton T1 partially relaxed using sequence
in Fig. 1a, t s 0.165 s, phase-shifted to make cellulose peaks face
upwards; Žc. proton T1r partially relaxed spectrum using sequence
in Fig. 1b, t s 22 ms; Žd. proton T2 partially relaxed using
sequence in Fig. 1c, t s 45 ms.
duced mobilisation w5,6,20x, some signals were still
present in the CPMAS spectrum ŽFig. 3a., indicating
that a substantial amount of pectin retained a significant level of static dipolar interactions with carbon.
Resolution improvement upon hydration is also
clearly observed, resulting from reduced conformational distribution. This is in accord with observations in cell wall materials of PB and CWC w20x.
Line-widths of pectin signals were not much broader
than those of cellulose, suggesting that those pectic
materials contributing to this spectrum have a relatively small distribution of conformations w20,45x.
The 1 H T1 relaxed subspectrum showed that the
long T1 component is associated with cellulose in
both crystalline and para-crystalline forms. No pectin
signal appeared in the long 1 H T1 subspectrum. This
suggests that spin diffusion between cellulose and
pectin under the conditions is not efficient in the
time scale of T1. However, the proton T1r partially
relaxed subspectrum ŽFig. 3c. showed resonances
from not only cellulose, but also from pectin. Since
T1 is an order of magnitude longer than T1r , it is not
possible for spin diffusion to be inefficient in the
time scale of T1 but efficient in the time scale of
T1r . Therefore, the appearance of pectin signals in
this subspectrum cannot be a result of spin diffusion.
The most likely explanation is that there are more
than two proton T1r processes, which converge in
values coincidentally rather than by spin-diffusion.
This sample not only showed multiple-component
processes for 1 H T1 and T1r , but also two components in proton T2 . The T2 ŽH. relaxed subspectrum
ŽFig. 3d, with a delay time of 45 ms. showed only
signals from pectin; therefore, some of the pectin
that appeared in CPMAS spectrum ŽFig. 3a. had a
longer T2 value than that of the cellulose shown in
Fig. 3a–c.
Since the number of protons from pectin and
cellulose is roughly the same and protons associated
with a short T2 Ž19.5 ms. accounted for 85% of the
total proton pool, some pectin must be associated
with the short T2 fraction. Assuming all cellulose
protons appeared only in the short T2 component,
about two-thirds Ž35% out of 50%. of the pectin
protons would be in the short T2 component. Given
the delay of 45 ms and assuming a Gaussian decay,
the short T2 component Ž19.5 ms. would be decayed
to less than 1%; therefore, this pectin fraction ap-
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
Fig. 4. PRISE spectra of dry CWC. Ža. Standard CPMAS spectrum; Žb. proton T1 partially relaxed using sequence in Fig. 1a,
t s 0.05 s, phase-shifted to make cellulose peaks face upwards;
Žc. difference spectrum, a–b; Žd. proton T1r partially relaxed
spectrum using sequence in Fig. 1b, t s15 ms; Že. difference
spectrum, a–d.
245
spectrum ŽFig. 4a., signals from both pectin and
cellulose were visible. Partially 1 H T1 relaxed subspectra ŽFig.4b., however, showed that the long T1
component was chiefly cellulose. A difference spectrum ŽFig. 4c. between fully relaxed and partially
relaxed spectra showed that the short T1 component
was mainly non-cellulose materials. Using the same
process with proton T1r partially relaxed spectra, the
long T1r component ŽFig. 4d. was associated with
cellulose signals and the shorter component ŽFig. 4e.
to the non-cellulose signals, even though the
signalrnoise ratio prohibited any quantitative interpretation.
Hydrating CWC with D 2 O to 88% led to changes
in both proton relaxation properties and 13 C CPMAS
spectra ŽFig. 5.. Proton T1 changed into a single
component; T1r remained as two components with
slight changes in the percentage contributions. T2
changed to a two-component relaxation process, in
which the fastest decaying component was Gaussian
and accounted for about two-thirds of all protons.
The long component was appropriately described
with an exponential decay function. The T1r partially
pearing in the T2 relaxed spectrum should have a
much longer T2 than 19.5 ms and can be associated
with the long T2 component in the proton measurement ŽTable 2.. This confirms the observation that
different pectin fractions have different molecular
mobilities w5,7,20x. In addition, the inefficient spin
diffusion between T1 components suggests that in
this simple mixture of pectin and cellulose both
chemical components are still behaving as separated
components such that the domain size is large compared to the distance spins can diffuse on a time
scale of T1 Ž; 40 nm w2x..
3.3. Cell wall materials of CWC
For D 2 O exchanged and lyophilised CWC, two
proton T1 and T1r components were observed when
the sample was spinning about 4 kHz at the magic
angle. These results are similar to the static measurements w6x. One T2 component was detected ŽTable 2.
as in the case of static measurements. In the CPMAS
Fig. 5. PRISE spectra of CWC containing 88% D 2 O. Ža. Standard
CPMAS spectrum; Žb. proton T1r partially relaxed using sequence
in Fig. 1b, t s 21 ms; Žc. difference spectrum, a–b; Žd. proton T2
partially relaxed using sequence in Fig. 1c, t s 46 ms.
246
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
relaxed ŽFig. 5b. and difference subspectra ŽFig. 5c.
showed that the long T1r component was associated
with cellulose whereas the short one was associated
with non-cellulose materials. A subspectrum ŽFig.
5d. obtained with 45 ms 1 H T2 relaxation before CP
showed that two T2 components were responsible for
transferring proton magnetisation to 13 C. The long T2
Ž103 ms. was mainly associated with pectin as indicated by resonances at 53 ŽCH 3 O. and 21 ppm
ŽCH 3 ., and hemicellulose Ž109 ppm. w20x, whereas
the short T2 Ž19.5 ms. was associated with both
cellulose and pectin, which was also implied by the
relative proton populations. T2 measured here represented protons involved in residual dipolar interactions with carbon under the experimental conditions
ŽMAS at 4 kHz.. Therefore, protons with weaker
static dipolar interactions compared to sample spinning rate Ž; 4 kHz. could not be observed in CPMAS experiments since such weak interactions would
be averaged with magic angle sample spinning. A
Fig. 7. PRISE spectra of PB containing 116% D 2 O. Ža. Standard
CPMAS spectrum; Žb. proton T1r partially relaxed using sequence
in Fig. 1b, t s 25 ms; Žc. difference spectrum, a–b; Žd. proton T2
partially relaxed using sequence in Fig. 1c, t s 40 ms; Že. SPEMAS spectrum with recycle delay of 1 s.
Fig. 6. PRISE spectra of dry PB. Ža. Standard CPMAS spectrum;
Žb. proton T1 partially relaxed using sequence in Fig. 1a, t s 0.16
s, phase-shifted to make cellulose peaks face upwards; Žc. difference spectrum, a–b; Žd. proton T1r partially relaxed using sequence in Fig. 1b, t s14.5 ms; Že. difference spectrum, a–d.
SPEMAS spectrum of CWC Ždata not shown. confirmed that there was substantial amount of pectin
mobilised by hydration w20x, having a rather short
13
C T1 Ž- 1 s.. As observed in the case of static
samples w7x, the 1 H T1 of the wet sample was a
single process whereas T1r was a two-component
process, in which cellulose relaxed much slower than
non-cellulose components. However, spin diffusion
should be much more efficient for protons in the dry
sample than in the wet one since hydration often
leads to mobilisation of biopolymers in the matrices
and thus weaker dipole–dipole interactions or less
efficient spin diffusion. Therefore, the presence of
multiple components for T1r and single component
of T1 for the wet sample implies that the uniformity
of T1 arises from a convergence of the relaxation
times rather than from mixing by spin diffusion w5,6x.
There is also a possibility that other mechanisms
H.-R. Tang et al.r Solid State Nuclear Magnetic Resonance 15 (2000) 239–248
might be contributors in spin–lattice relaxation in
addition to spin diffusion.
3.4. PB cell wall materials
Dry PB showed both signals from cellulose and
pectin in its CPMAS spectrum ŽFig. 6a. and two
components in its proton T1 and T1r , but effectively
a single component in T2 ŽTable 1. as in the case of
this sample without spinning w5,6x. PRISE experiments showed that the long T1 component ŽFig. 6b.
was associated with cellulose and a fraction of pectin
while the short T1 ŽFig. 6c. was associated with
chiefly pectin. This implies that there are two fractions of pectic materials having different proton T1
values, which has been observed and discussed elsewhere w20x. The pectin fraction having the same
proton T1 as cellulose must be either spatially close
to cellulose microfibrils so that spin diffusion between protons in them is fast on the time scale of 1 H
T1 , or coincidentally having the same T1 value.
The T1r relaxed ŽFig. 6d. and difference spectra
ŽFig. 6e. showed that cellulose is associated with the
long component of T1r and pectin with the short one.
Hydration of PB to 116% with D 2 O resulted in a
substantial loss of pectin signals in CPMAS spectrum ŽFig. 7a. due to mobilisation w20x of pectin by
water w5,6,30x. A single proton T1 was observed
while two components were observed for T1r and T2
ŽTable 2.. The long T1r component ŽFig. 7b. was
associated with cellulose whereas the short one ŽFig.
7c. was associated with non-cellulose components
Žmostly pectin.. The 1 H T2 partially relaxed subspectra ŽFig. 7d. showed that pectin had longer proton T2
than that of cellulose. A SPEMAS w20x spectrum
obtained with a recycle delay of 1s is shown in Fig.
7e, in which signals of pectin Ž170–174, 98–104,
60–84, 53, 18 ppm. w20x and hemicellulose Ž106–
110, 21 ppm. w20x are both visible. No resonance can
be seen for cellulose.
To sum up, PRISE experiments offer a great
opportunity to enable one to relate structural characteristics with the molecular dynamics properties in
plant cell walls. Therefore, they are powerful techniques to probe heterogeneity of the plant cell walls
or other heterogeneous systems. Although this paper
only described examples with two relaxation components, one would expect that heterogeneous systems
247
with more than two domains can be investigated
similarly using the appropriate algorithm to record
subspectra w 19,46,47 x , provided that good
signalrnoise ratio can be obtained. In plant cell
walls Ždry or wet., cellulose is always associated
with the long relaxation components of T1 and T1r
whereas pectin is associated with the shorter one. In
addition, cellulose is always associated with the
fast-relaxed T2 component. This is consistent with
the observation that cellulose is ‘‘rigid’’ whereas the
‘‘rigidity’’ of the pectin is highly dependent upon
the hydration levels.
Acknowledgements
Competitive Strategic Grant from BBSRC is acknowledged. We thank Dr. Annie Ng at Institute of
Food Research for providing cell wall materials of
potatoes and Chinese water chestnuts, and Mr. Reg
Wilson for his suggestions on the manuscript. We
also thank an anonymous referee for hisrher constructive criticism.
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