Journal of Chromatography A, 1128 (2006) 90–96
Resolution of complex monosaccharide mixtures from plant cell wall
isolates by high pH anion exchange chromatography
Heather A. Currie, Carole C. Perry ∗
Biomolecular and Materials Interface Research Group, School of Biomedical and Natural Sciences,
Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
Received 8 April 2006; received in revised form 2 June 2006; accepted 13 June 2006
Available online 3 July 2006
Abstract
The use of high pH anion exchange chromatography combined with pulsed amperometric detection has been established as an effective and
sensitive method for the separation, detection and quantification of monosaccharides from a wide range of sources. However, careful examination
of the separation conditions required is necessary to ensure that a complete monosaccharide profile can be determined from structures such as
the plant cell wall which is a complex network of both neutral and charged polysaccharides. This study has investigated the optimal conditions
required for the analysis of such a challenging mixture, including both the stationary and mobile phase minimising co-elution and reducing method
complexity. The preferred methods have been used to successfully identify and quantify the monosaccharide components of a selected extract from
the plant cell wall of the primitive higher plant Equisetum arvense.
© 2006 Elsevier B.V. All rights reserved.
Keywords: High pH anion exchange chromatography; Pulsed amperometric detection; Carbohydrates; Plant cell wall
1. Introduction
The plant cell wall is a highly complex three dimensional
network of interwoven polysaccharide chains embedded in a
gel matrix of galacturonic acid rich polysaccharides connected
by calcium bridges. This network also contains many structural proteins and glycoproteins combined with and partially
cross linked by phenolic substances [1,2]. As a result of this
intricate structure, the identification and quantification of the
individual components can be both problematic and time consuming.
The use of high pH anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) has
been used for a number of years for the detection and quantification of both mono and oligosaccharides. The use of this
technique is advantageous due to the high levels of detection
possible (down to picomolar levels). Also, it allows the determination of intact monosaccharides without pre or post column
∗
Corresponding author. Tel.: +44 115 8486695.
E-mail address: [email protected] (C.C. Perry).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2006.06.045
derivatisation, decreasing the time of analysis and eliminating
a decrease in recovery due to incomplete derivatisation [3–6].
The separated mono or oligosaccharides are therefore recoverable for further examination, vital when sample size is restricted.
The use of this technique for the analysis of plant monosaccharide composition has been examined by a number of researchers
with varied success largely due to the complexity of the material.
More recently, a comparison of the identification and quantification of monosaccharides by HPEAC-PAD and HPEAC coupled
with electrospray mass spectrometry found HPEAC-PAD to
be the most accurate routine method for the quantification of
monosaccharides [7].
Several groups have proposed separate methods for the separation of neutral and charged monosaccharides due to the much
stronger elution conditions required to remove the charged
species from the column [8,9]. However, running both species
together is possible and indeed preferable. The use of separate
runs for different monosaccharides without running several
standards which can be compared in both runs can make validation of the methods problematic. A single or combination of
methods analyzing the same components allows the researcher
to corroborate the efficiency and accuracy of the methods used.
H.A. Currie, C.C. Perry / J. Chromatogr. A 1128 (2006) 90–96
In order to examine the uronic acids, a method allowing the accurate separation of the neutral monosaccharides is required and
following this separation a rapidly increasing gradient of sodium
hydroxide and sodium acetate will elute the more charged
species.
Methods for the separation of plant derived monosaccharides
combining the neutral and uronic acid samples have been examined by a number of researchers [6,10,11]. These researchers
have had mixed results with the resolution of the individual monosaccharides. Work carried out by De Ruiter et al.
[10] utilised the original CarboPac PA1 column and using a
triple waveform for PAD detection reported a good separation of all the monosaccharides over a long run time. However, no full trace separation was shown so the sensitivity at
baseline level could not be examined. The original PA1 column was then improved upon to give the CarboPac PA10 by
increasing the degree of resin cross linking and decreasing
the bead size. The chemical compatibility of the resin with
HPLC solvents was also greatly increased from 2% to 90%
of common HPLC solvents. These changes in resin composition increased the sensitivity, and gave superior selectivity
over the original PA1 column as evidenced by Salvador et
al. [11] on comparison of the two columns during a study of
cell wall monosaccharides of cassava and potato. This study
utilised a very low gradient of only 0.5 mM sodium hydroxide to elute 6 neutral monosaccharides and this increased to
incorporate 125 mM sodium acetate in 200 mM sodium hydroxide to elute galacturonic acid. d-glucosamine and glucuronic
acid were not investigated. A post column addition of 300 mM
sodium hydroxide was used to aid detection. The separation
of the individual monosaccharides was not shown and therefore it is not possible to comment on the accuracy of this
method. Most work on developing methods for the separation of complex monosaccharide mixtures by HPAEC-PAD has
been carried out using the CarboPac PA1 or PA10 columns.
However, a new column has since been released which utilises
the same column matrix as the PA10 but with decreased substrate diameter and functionalised microbead size allowing
for improved column performance and chemical compatibility over the earlier PA10. This new column has been utilised
recently by Guignard et al. [7] to separate the monosaccharides present in an ethanol soluble extract from the plant cell
wall prior to analysis by mass spectrometry. The PA20 has
also been utilised by Eberendu et al. [12] to study the separation of a number of monosaccharides derived from glyconutritional products. This study achieved separation of a
number of monosaccharides but again incorporated the use of
post column addition of sodium hydroxide to increase detector
response.
This paper describes the optimisation of the separation of ten
monosaccharides from plants by comparing two column types
CarboPac PA10 and the newest column the PA20. The advantages and disadvantages of each column are discussed with the
optimum methods for each column described. The two different
stationery phases were then compared for their ability to separate a plant cell wall extract from the primitive higher plant
Equisetum arvense.
91
2. Experimental
2.1. High pH anion exchange chromatography with pulsed
amperometric detection for the identification and
quantification of monosaccharides
The individual monosaccharides of cell wall fractions were
analysed by the use of a Dionex BioLC system composed of
an electrochemical detector (ED50), gradient pump (GP50) and
injector system (LC20). Data was collected and analysed using
Chromeleon software (version 6.5) and the detector utilised a
pulsed quadruple waveform for the detection of carbohydrates
as developed by Rocklin et al. [13], Table 1. For each injection a
25 ␮l volume was used coupling 20 ␮l of sample with a further
5 ␮l of a 6 mM solution of 2-deoxy-d galactose as an internal
standard.
Development of a method for the efficient separation and
quantification of plant cell wall monosaccharides was carried out
examining a range of sodium hydroxide and sodium acetate concentrations. The eluents used in the separation of the monosaccharides l-fucose, l-arabinose, l-rhamnose, d-galactose, dglucosamine, d-glucose, d-xylose, d-mannose, d-galacturonic
acid and d-glucuronic acid were as follows, A-150 mM NaOH,
B-1 M CH3 COONa in 150 mM NaOH and C-HPLC grade water.
A borate trap was added to the line prior to injection to eliminate trace levels of borate ions. Examination of the CarboPac
PA20 column was carried out using a number of different elution profiles and these are shown in Table 2. After each method
the column was washed with 10 ml of 150 mM NaOH and then
regenerated with 10 ml of the starting solutions.
2.2. Cell wall extraction from E. arvense
The plant cell wall was extracted using ice cold 70% ethanol
and vortexed and cooled to 0 ◦ C at least five times. The mixture
was then stirred at 0 ◦ C for 5 h before filtration under vacuum.
The insoluble residue was washed with further aliquots of
70% ethanol. The alcohol insoluble residue was stirred in a
50 mM solution of the aqueous chelating agent CDTA pH
7.0 at room temperature for 16 h. The insoluble material was
removed by centrifugation and extracted again by this method.
The supernatant was retained and dialysed using 3,500 MW
cut off membrane for the removal of the CDTA. This process
was then repeated with the insoluble material to generate a
second extract. The CDTA insoluble material was extracted
Table 1
The quadruple waveform utilised for the detection of carbohydrates
Time (s)
Potential (V)
0.00
0.2
0.4
0.41
0.42
0.43
0.44
0.5
+0.1
+0.1
+0.1
−0.2
−0.2
+0.6
−0.1
−0.1
Integration
Begin
End
92
H.A. Currie, C.C. Perry / J. Chromatogr. A 1128 (2006) 90–96
Table 2
Elution profiles examined using the CarboPac PA20 to separate monosaccharides of the plant cell wall
Elution profile
A
B
C
D
Isocratic 1.5 mM NaOH with a gradient increasing from 2% to 17% of 1 M sodium acetate and 150 mM sodium hydroxide between 20 min and 40 min
Isocratic 4.5 mM NaOH with a gradient increasing from 2% to 17% of 1 M sodium acetate and 150 mM sodium hydroxide between 20 min and 40 min
Isocratic 6 mM NaOH with a gradient increasing from 2% to 17% of 1 M sodium acetate and 150 mM sodium hydroxide between 20 min and 40 min
Isocratic 9 mM NaOH with a gradient increasing from 2% to 17% of 1 M sodium acetate and 150 mM sodium hydroxide between 20 min and 40 min
further with a mixture of 50 mM sodium carbonate and 20 mM
sodium borohydride. This suspension was stirred for 16 h at
4 ◦ C and the soluble extract removed by centrifugation and
the supernatant collected and dialysed using 3,500 MW cut off
membrane to a conductivity ≤1 ␮S and the retentate freeze
dried (VirTis) [1]. The extracted samples were hydrolysed for
the release of monosaccharides with 2 M trifluoroacetic acid
for 1 h.
3. Results
3.1. Separation of monosaccharides from the plant cell
wall using the PA20 column
The CarboPac PA20 column was initially tested for its ability to separate the complex mixture of monosaccharides that
are generally thought to be present in the plant cell wall which
includes l-fucose, l-arabinose, l-rhamnose, d-galactose, dglucosamine, d-glucose, d-xylose, d-mannose and the uronic
acids, d-galacturonic acid and d-glucuronic acid. This complex mixture poses several difficulties due to the similarities in
structure and charge of the molecules. In order to determine
the elution conditions resulting in baseline separation of the
monosaccharides, a number of different elution profiles and flow
rates were examined with the chromatographic traces obtained
shown in Fig. 1. The initial elution conditions tested utilised the
low level isocratic sodium hydroxide concentration 1.5 mM to
separate the neutral monosaccharides at a flow rate of 0.5 ml/min
(Fig. 1A). From this trace it is possible to observe a clear baseline to baseline separation for most of the monosaccharides with
the exception of d-galactose and d-glucosamine which co-elute,
with galactose present as a shoulder on the glucosamine peak.
The charged uronic acids are clearly resolved by the introduction
of an increasing sodium acetate gradient. The use of a 1.5 mM
sodium hydroxide isocratic solution is the lowest level suitable
for monosaccharide analysis with a pH of 11.9, PAD detection
with gold electrodes is most suited to pH ≥12 [14]. As this low
concentration method was not wholly suitable for the separa-
Fig. 1. Optimisation of the separation of monosaccharides on the PA20 column. The monosaccharides are indicated as follows (1) fucose; (2) 2 deoxy d-galactose;
(3) arabinose; (4) rhamnose; (5) galactose; (6) glucosamine; (7) glucose; (8) xylose; (9) mannose; (10) galacturonic acid; (11) glucuronic acid. All method utilised
an isocratic elution for the first 20 min followed by the introduction of sodium acetate with sodium hydroxide. The isocratic elution is as follows (A) 1.5 mM sodium
hydroxide; (B) 4.5 mM sodium hydroxide; (C) 6 mM sodium hydroxide; (D) 9 mM sodium hydroxide.
H.A. Currie, C.C. Perry / J. Chromatogr. A 1128 (2006) 90–96
tion of galactose and glucosamine it was necessary to investigate
higher sodium hydroxide gradients.
An increase in sodium hydroxide concentration to 4.5 mM did
start to show a slight separation of galactose and glucosamine at
a retention time of 10 min, but now the increase in charge due to
the higher sodium hydroxide concentration lead to glucosamine
starting to elute slightly earlier than galactose with a small
degree of peak splitting (Fig. 1B). However, this alteration of
sodium hydroxide concentration also resulted in the co-elution
of l-arabinose and l-rhamnose. The time between the d-xylose
(8) and d-mannose (9) peaks is also significantly reduced. Further increase of the sodium hydroxide concentration to 6 mM
(Fig. 1C) again demonstrated the co-elution of l-arabinose and
l-rhamnose and there is a reduction in the degree of separation of d-xylose and d-mannose. There is however an increase
in separation between d-glucosamine and d-galactose but complete separation of these monosaccharides did not occur until a
concentration of 9 mM was used (Fig. 1D). This corresponded
with near complete co-elution of d-xylose and d-mannose. These
results demonstrate that the optimum conditions for the analysis
of monosaccharides from the plant cell wall would be an isocratic solution of 1.5 mM sodium hydroxide with sodium acetate
introduced after separation of the neutral sugars with an increasing gradient from 20 mM to 250 mM. The separation of galactose
and glucosamine when required can be examined with a higher
isocratic sodium hydroxide concentration of 9 mM. The reproducibility of these results relative to an internal standard are
shown in Table 3.
93
Fig. 2. Separation of a monosaccharide mixture with using the CarboPac PA10
column.
duction of a sodium acetate gradient from 20 to 200 mM. This
separation is shown in Fig. 2 and the reproducibility of this
method was examined with the retention times compared to an
internal standard, Table 4. The relative retention times for the
uronic acids did show a larger degree of variation in retention
time in relation to the standard but little variation in the actual
time retained on the column so these values are also shown.
3.3. Analysis of cell wall extract from E. arvense by the
optimized methods
The older PA10 column was also analysed for its ability to
separate the complex mixture of monosaccharides found in the
plant cell wall. Of the methods examined the most suitable was
found to also utilise an isocratic solution of 1.5 mM sodium
hydroxide with a flow rate of 0.5 ml/min for the separation of
l-arabinose, l-rhamnose, d-galactose, d-glucosamine and dglucose, with an increase to 4.5 mM to separate d-xylose and
d-mannose. Separation of the uronic acids required the intro-
Analysis of a hydrolysed cell wall extract, anticipated to contain both neutral and charged monosaccharides from E. arvense
was carried out on both columns utilising the optimised conditions for each and the results are shown in Fig. 3. The separation
achieved by the PA10 column is shown in Fig. 3A allowing the
identification of most of the main peaks present such as arabinose and glucose. However the greater degree in retention time
variation, as shown in Table 4 demonstrates a possible disadvantage of the PA10. The large peak at 50 min is most likely to be
galactose; however the relative retention time of 2.32 falls just
outside the limits shown in Table 4. This also occurs with the
Table 3
The relative retention times of the individual monosaccharides separated with
the CarboPac PA20 column demonstrating the repeatability of the separations
Table 4
The relative retention times of the monosaccharides demonstrating the repeatability of method F using the CarboPac PA10 column
3.2. Separation of monosaccharides from the plant cell
wall using the PA10 column
Mean relative retention timea
Mean relative retention timea
Fucose
Arabinose
Rhamnose
Glucosamine
Galactose
Glucose
Xylose
Mannose
Galacturonic acid
Glucuronic acid
0.77
1.80
1.89
1.85
2.03
2.77
3.37
3.75
6.76
7.25
±
±
±
±
±
±
±
±
±
±
0.01
0.02
0.02
0.03
0.04
0.03
0.03
0.03
0.2
0.2
Method D was utilised for the separation of glucosamine and galactose, all other
monosaccharides were analysed utilising method A.
a Values calculated using a minimum of 11 samples.
Fucose
Arabinose
Rhamnose
Galactose
Glucosamine
Glucose
Xylose
Mannose
Galacturonic acid
Glucuronic acid
0.73
1.75
1.99
2.23
2.56
2.78
3.31
3.57
4.76
5.97
±
±
±
±
±
±
±
±
±
±
0.01
0.02
0.02
0.07
0.04
0.06
0.03
0.14
0.54
0.56
Mean retention timea
93.11 ± 0.28
97.08 ± 0.29
The mean retention times are shown for the charged uronic acids and demonstrate
greater reliability for identification.
a Values calculated using a minimum of 6 samples.
94
H.A. Currie, C.C. Perry / J. Chromatogr. A 1128 (2006) 90–96
Table 5
Composition of a plant cell wall extract determined by separation on the PA20
column after hydrolysis with 2 M TFA for 1 h
nmol/mg of extract
Fucose
Arabinose
Rhamnose
Glucosamine
Galactose
Glucose
Xylose
Mannose
Galacturonic acid
Glucuronic acid
0.89
133.33
34.58
12.15
171.90
63.64
3.3
16.30
40.32
23.03
of the separation is greater allowing a more confident identification of the individual monosaccharides from the hydrolysed
sample. Due to the greater accuracy of monosaccharide identification and swiftness of separation, the PA20 column was used as
means of quantifying the monosaccharides present in the hydrolysed plant cell wall sample.
3.4. Quantification of monosaccharides separated by the
PA20 column
Analysis of the separation of monosaccharide mixtures of
varying concentrations demonstrated the ability of this column
to be utilised as a means of quantitative analysis for hydrolysed
oligo- or polysaccharides. The calibration data for a selection
of the monosaccharides is shown in Fig. 4 demonstrating the
accuracy of this method. The results generated from this study
allowed the quantification of the monosaccharides present in a
hydrolysed sample of the plant cell wall, Table 5. From this data
it is apparent that the sample is predominantly composed of arabinose and galactose which could indicate the sample contained
glycosylated arabinogalactan proteins. However, it is thought
that the conditions employed did not yield a complete hydrolysis
due to the presence of a number of unidentified peaks which are
likely to be di- or oligosaccharides. The conditions required for
the complete hydrolysis of plant cell wall oligosaccharides still
requires some investigation to determine the conditions required
to release all the monosaccharides without degradation of those
that are less resistant to the acidic conditions.
Fig. 3. Comparison of separation of a plant cell wall extract by (A) PA10 column;
(B) PA20 column; (C) PA20 column for the separation of Glucosamine (6) and
Galactose (5).
peak at 62 min (relative retention time 2.85) which is likely to
be glucose. This could result in some ambiguity when identifying monosaccharides from an unknown composition. Another
negative aspect of analysing samples with the PA10 column is
the length of time, over 2 h, to run each sample.
Analysis of a hydrolysed plant cell wall sample utilising the
PA20 column is shown in Fig. 3B and C. Despite the need to analyse the sample by two different methods, the complete analysis
time is still shorter than the previous method. The repeatability
4. Discussion
The separation of the complex mixture of monosaccharides
found in the plant cell wall presents researchers with a challenging chromatographic problem. The separation of monosaccharides by use of high pH anion chromatography with pulsed
amperometric detection is a valuable technique for the separation, identification and quantification of monosaccharides.
Small quantities, down to picomole level can be observed using
this technique and derivatisation of the monosaccharides is not
required as in mass spectrometry and gas chromatography allowing their recovery after separation [3]. HPAEC-PAD allows for
the differentiation of monosaccharides that are chemically very
H.A. Currie, C.C. Perry / J. Chromatogr. A 1128 (2006) 90–96
95
Fig. 4. Calibration data for monosaccharides analysed on the PA20 column. (A) Glucose; (B) rhamnose; (C) galacturonic acid; (D) glucuronic acid; (E) glucosamine;
(F) xylose.
similar and their quantification in relation to the PAD response.
Separation is achieved through the exploitation of the weakly
acidic nature of monosaccharide hydroxyl groups at high pH
(>12). The monosaccharides can then be resolved as oxyanions
utilising the slight variations in the pKa values of the monosaccharides [4,5].
The PA10 column was capable of achieving a full separation of a mixture of the monosaccharides examined but did have
several disadvantages. The run was prohibitively long with the
final time being 130 min after cleaning and regeneration due to
the low flow rate required. The analysis of an actual hydrolysed plant cell wall was more difficult with the PA10 due to
the greater degree of variation in relative retention times. Two
peaks occurring in this trace at approximately 50 and 60 min are
assumed to be galactose and glucose respectively due to their
presence in the PA20 trace of the same sample. However, the
actual relative retention times were slightly different from the
values listed in Table 3. The limited solvent compatibility of the
PA10 was also found to be restrictive. This column although
being compatible with 90% of common HLPC solvents cannot
be used with pure water as this causes considerable alteration
to the column properties. The newer PA20 column has no such
problems allowing the use of water for simple operations such
as monitoring pressure and pump operation. The newer PA20
column was capable of separating all the monosaccharides to
baseline with the exception of d-glucosamine and d-galactose
which showed a degree of co-elution. This problem however,
was easily overcome with the use of the 9 mM isocratic elution
solely for the neutral monosaccharides which would ascertain if
both of these monosaccharides are present. If this combination
is found in samples the use of both the 1.5 mM and 9 mM methods, calibrated individually is still less time consuming than the
method required for the PA10. Those sugars that are baseline
separated with both elution profiles, such as l-fucose, can be
used to confirm continuity between the two methods. The superiority of the PA20 column was clearly demonstrated by the
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H.A. Currie, C.C. Perry / J. Chromatogr. A 1128 (2006) 90–96
analysis of a plant cell wall extract which allowed the straightforward identification of the monosaccharides by their retention
time relative to the standard. Also observed in this trace was
the clear determination of as yet unidentified sugars. This separation therefore allows the recovery of the unidentified peaks
which could be analysed in their native form by a variety of
methods due to HPAEC-PAD not requiring pre-derivatisation of
the monosaccharides.
This research has resulted in optimized methods for the separation of monosaccharides found in the plant cell wall using
the PA20 column. Previous work by other research groups
have analysed the plant cell wall monosaccharides however,
these have often involved more complex means of separation
and detection such as the need for post column addition of
base for increased sensitivity [9,12,15]. The detection of some
monosaccharides, for example glucosamine has been omitted
from previous studies [9,11,16,17]. While difficulties can arise
in elucidating this monosaccharide from galactose the determination of its presence is vital as it occurs due to nucleophilic acyl
substitution of N-acetyl-glucosamine during acid hydrolysis. NAcetyl-glucosamine is required for N linked glycosylation of
proteins and is therefore an important structural feature that can
be hypothesized from monosaccharide composition.
5. Conclusion
This study has examined different methods for the optimal
separation of complex monosaccharide mixtures by high pH
anion exchange chromatography coupled with pulsed amperometric detection. Analysis of a mixture of ten different neutral
and charged monosaccharides commonly found in the plant cell
wall was carried out varying both the stationary and mobile
phases. An examination of two columns was carried out and
these were found to be capable of resolving a mixture of
monosaccharides without the need for derivatisation or post
column addition of base. However, separation using the PA20
column resulted in much shorter run times and greater resolution
of the peaks and this was proven with the separation of a hydrolysed plant cell wall extract. The separation of d-galactose and
d-glucosamine was possible with the PA20 utilizing a separate
method with higher alkali concentration and this again required
a short run time. The separation of a sample of hydrolysed plant
cell wall by the PA20 column allowed the clear identification of
the monosaccharides allowing their quantification. The methods
proposed herein combine reliable and reproducible detection and
separation and as such simplify the process of identifying and
quantifying the monosaccharide components of complex biological materials such as the plant cell wall.
Acknowledgement
The financial support of the AFOSR is gratefully acknowledged.
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