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Carbohydrate analysis of plant materials with uronic


acid-containing polysaccharidesA comparison between
different hydrolysis and subsequent chromatographic
analytical techniques
Stefan Willfr a, , Andrey Pranovich a , Tarja Tamminen b , Juergen Puls c ,
Christiane Laine d , Anna Suurnkki b , Bodo Saake c , Kati Uotila b ,
Helena Simolin b , Jarl Hemming a , Bjarne Holmbom a
a

Process Chemistry Centre, bo Akademi University, Porthansgatan 3, FI-20500 Turku, Finland


VTT Technical Research Centre of Finland, Tietotie 2, Espoo, FI-02044 VTT, Finland
c vTI-Institute for Wood Technology and Biology, Leuschnerstr. 91, D-21031 Hamburg, Germany
d Oy Keskuslaboratorio - Centrallaboratorium Ab, Tekniikantie 2, 02150 Espoo, Finland
b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Acid hydrolysis, acid methanolysis, and enzymatic hydrolysis were compared for depoly-

Received 26 March 2008

merization of ve different plant materials containing uronic acids. The analyzed plant

Received in revised form

materials were oat spelt, wheat straw, spruce thermomechanical pulp, aspen stemwood,

7 November 2008

and totally chlorine-free (TCF) bleached hardwood kraft pulp. Furthermore, GC (using both

Accepted 10 November 2008

HP-1 and HP-5 capillary columns and FID and MSD detectors), HPAEC-PAD, and HPAECBorate techniques were compared for subsequent analysis of the released monosaccharides.
It was shown that acid methanolysis combined with GC analysis is a convenient method

Keywords:

for obtaining the sugar unit composition and amount of non-crystalline polysaccharides in

Acid methanolysis

different plant materials. The methanolysis method was generally superior to the hydrol-

Acid hydrolysis

ysis method for xylan- and uronic acid-containing samples. However, acid and enzymatic

Enzymatic hydrolysis

hydrolysis showed the highest recoveries for bleached chemical pulp samples. Acid hydrol-

GC-FID

ysis is also required for crystalline polysaccharides, but the strong acid conditions evidently

GCMS

lead to degradation of labile sugars. The plant methanolysates were not suitable as such for

HPAEC-PAD

analysis on an HPAEC-PAD system. For analysis of the total amount of sugar units, hence

HPAEC-Borate

including cellulose, other non-crystalline hemicelluloses, and pectins, a combination of the


methanolysis and hydrolysis methods is recommended.
2008 Elsevier B.V. All rights reserved.

1.

Introduction

Cellulose, hemicelluloses, starch, and pectins account for


most of the biomass in annual and perennial plants.

Furthermore, higher plants contain various amounts of different uronic acid-containing hemicelluloses and pectins.
The cell wall polysaccharides mainly consist of different
pentoses (-D-xylose, -L-arabinose), hexoses (-D-glucose,

Corresponding author at: Laboratory of Wood and Paper Chemistry, Process Chemistry Centre, bo Akademi University, Porthansgatan
3, FI-20500 Turku, Finland. Tel.: +358 40 5047904; fax: +358 2 215 4868.
E-mail address: swillfor@abo. (S. Willfr).
0926-6690/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2008.11.003

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i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

Table 1 Details of each depolymerization method used in the study. Five determinations were made per sample.
Acid hydrolysis

Acid methanolysis

Enzymatic hydrolysis

Sample amount

100 mg

10 mg

500 mg

Depolymerization
reagent

Pre-hydrolysis: 1 mL
70% H2 SO4 (VTT, BFH) or
1 mL 72% H2 SO4 (KCL)
Hydrolysis: addition of
28 mL ultra-pure water
Deoxy-glucose and
fucose, added after the
hydrolysis
Pre-hydrolysis in water
bath: 30 C, 1 h
Hydrolysis in autoclave:
120 C, 50 min (VTT,
BFH) or 60 min (KCL)
No calibration for the
hydrolysis

2 mL 2 M HCl in
anhydrous
MeOH

Enzyme mixture: 50 FPU/g, 500010,000 nkat/g major


endo-hydrolases (endo-1,4-b-glucanase, endo-1,4-b-xylanase
and endo-1,4-b-mannanase), 10002000 nkat/g b-glucosidase,
100500 nkat/g a-arabinosidase and a-galactosidase and
50200 nkat/g b-mannosidase
Fucose, added after the hydrolysis

Internal standard

Reaction
conditions

Calibration

0.1 mg sorbitol in MeOH,


added after the
methanolysis
100 C, 5 h
(ABO) or 3 h
(KCL)

Methanolysis of
separate samples with
0.1 mg of each sugara in
MeOH (ABO) or 0.2 mg of
Xyl, Man, Glc and 0.1 mg
of the other sugars in
MeOH (KCL)

40 C, 48 h, pH 5 (sodium acetate buffer),


then boiling for 5 min to deactivate
enzymes

No calibration for the hydrolysis

Except 4-O-MeGlcA.

-D-mannose, -D-galactose), uronic acids (-D-glucuronic


acid, -D-galacturonic acid, -D-4-O-methylglucuronic acid),
or small amounts of deoxy-hexoses (-L-rhamnose, -Lfucose) (Sjstrm, 1993; Whistler, 1993; Waldron and Faulds,
2007). Different homo- (e.g. cellulose) or heteropolysaccharides (e.g. glucuronoarabinoxylans) are built up of anhydrosugar residues covalently linked by glycosidic linkages. The
polysaccharides can be entangled to or even covalently bound
to lignin, proteins, or extractives in the cell wall matrix.
Accurate analysis of the carbohydrate composition and
amount in higher plants, thus including uronic acidcontaining polysaccharides, is often desired for monitoring
process control and product quality parameters and for studying biological aspects in food and non-food applications. The
analysis of bre charge in the elds of pulp and paper is
also important for process and product control purposes.
Consequently, the cell wall polysaccharides rst need to be
depolymerized into their constituent sugar residue, which
commonly is done by acid-catalyzed hydrolysis of glycosides
(BeMiller, 1967; Tappi T 249 cm-85, 1985; De Ruiter et al.,
1992; Vuorinen and Aln, 1999; Kamerling and Gerwig, 2007).
However, this procedure is not that straightforward even for
isolated, lignin-free polysaccharides, since the susceptibility
of different glycosidic linkages towards acid hydrolysis varies.
Uronic acids are also often involved in very acid-resistant
glycosidic linkages (De Ruiter et al., 1992; Dahlman et al.,
2000; Tenkanen et al., 1995). The cell walls in higher plants
are usually lignied and contain highly ordered (crystalline)
cellulose, meaning that the hydrolytic conditions need to
be more powerful to fully depolymerize the polysaccharides
in the sample matrix. Consequently, the analysis result of
hydrolyzed cell wall polysaccharides will be a compromise
between total depolymerization and a minimum of degradation of liberated monosaccharides. Other methods used
for the depolymerization step of plant polysaccharides are

acid methanolysis (De Ruiter et al., 1992; Bleton et al., 1996;


Sundberg et al., 1996; Vuorinen and Aln, 1999; Bertaud et al.,
2002; Mejanelle et al., 2002; Kamerling and Gerwig, 2007) and
enzymatic hydrolysis (Vuorinen and Aln, 1999; Dahlman et
al., 2000; Kamerling and Gerwig, 2007). It has been discovered
that acid methanolysis depolymerizes exclusively hemicelluloses very efciently when applied directly on wood samples,
without any pretreatment such as delignication. However,
the method requires dried samples, so freeze-drying is recommended. Acid methanolysis has recently successfully been
applied directly on wood of different tree species (Willfr et
al., 2005a, 2005b) and has also been shown to be sufcient
even for small sample amounts down to 0.2 mg wood (Marga
et al., 1995; Pranovich et al., 2006), while enzymatic hydrolysis of polysaccharides works well only on delignied samples
(Dahlman et al., 2000).
Monosaccharides in hydrolysates or methanolysates are
normally analyzed by gas chromatography with ame ionization detection (GC-FID) and/or gas chromatography with
mass spectrometric detection (GCMS) (Bleton et al., 1996;
Sundberg et al., 1996; Dahlman et al., 2000; Mejanelle et
al., 2002; Kamerling and Gerwig, 2007), high-performance
anion-exchange chromatography with pulsed amperometric
detection (HPAEC-PAD) (De Ruiter et al., 1992; Vuorinen and
Aln, 1999; Kamerling and Gerwig, 2007), high-performance
borate-complex anion-exchange chromatography with spectrophotometric detection at 560 nm (HPAEC-Borate) (Sinner et
al., 1975; Puls, 1993) or capillary electrophoresis (CE) (Dahlman
et al., 2000). GC is an established method with superb
resolution and high sensitivity which, however, requires
derivatization prior to analysis. HPAEC and CE can be performed without derivatization, but then suffers from relatively
poor separation and sensitivity. In HPAEC-PAD, as well in
HPAEC-Borate, neutral and acid sugars may be eluted in one
single run by appropriate choice of the gradient.

i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

Table 2 Participating laboratories, depolymerization,


and analytical methods used in the study.
VTTa
Acid hydrolysis
Acid methanolysis
Enzymatic hydrolysis
GC (HP-1/HP-5)
HPAEC-PAD
HPAEC-Borate
a
b

c
d

BFHb
e

KCLc

ABOd

VTT Technical Research Centre of Finland, Espoo, Finland.


vTI-Institute for Wood Technology and Biology, Hamburg, Germany.
Oy Keskuslaboratorio - Centrallaboratorium Ab, Espoo, Finland.
Process Chemistry Centre, bo Akademi University, Turku, Finland.
Also one larger hydrolysis batch of each sample was distributed
to the other participating laboratories for carbohydrate analysis.

Various methods for depolymerization and subsequent


analysis of released sugar residues in annual and perennial
plants have been reported in the literature, but few critical
comparisons are found. Nevertheless, the hydrolytic procedure and the subsequent analysis have been pointed out
as problematic (Puls, 1993; Chum et al., 1994; Jacobs, 2003;
Jacobs et al., 2003). Especially the compromise between incomplete hydrolysis and sugar destruction, as well as differences
in equipment and calibration between laboratories are indicated. Thus a comparative study to compare commonly used
methods for carbohydrate analysis of solid plant materials,
especially with uronic acid-containing polysaccharides, was
indeed needed. In the present paper, acid hydrolysis, acid
methanolysis, and enzymatic hydrolysis (Tables 1 and 2) were
compared for depolymerization of ve different plant materials containing uronic acids. The analyzed plant materials were
oat spelt, wheat straw, spruce thermomechanical pulp, aspen
stemwood, and totally chlorine-free (TCF) bleached hardwood
kraft pulp. Furthermore, GC (HP-1 and HP-5 capillary columns,
MS and FID detectors), HPAEC-PAD, and HPAEC-Borate (anion
exchange chromatography) techniques were compared for
subsequent analysis of released sugar residues.

2.

Experimental

2.1.

Materials

using a Fritsch Pulverisette mill (Fritsch GmbH, Germany)


equipped with a 0.5 mm sieve. All samples were freeze-dried
before distribution to respective laboratory.

2.2.

Acid hydrolysis

Acid hydrolysis (Table 1) was performed according to the


method originally described by Saeman et al. (1954). In short,
100 mg of freeze-dried material was weighted into a hydrolysis
tube, 1 mL of 70% (VTT and BFH, see Table 2 for explanation) or
72% (KCL, Table 2) (w/w) sulphuric acid was added and samples
were pre-hydrolyzed at 30 C in water bath for 1 h. The samples
were transferred into a 50 mL volumetric ask with addition of
28 mL of ultra-pure water. Flasks were sealed with aluminum
foil and autoclaved for 50 min (VTT and BFH) or 60 min (KCL)
at 120 C. After hydrolysis, the samples were cooled and ltered with 0.45 m GHP lters. The hydrolysis was performed
as ve replicate analyses. Hydrolyzed samples were diluted
with ultra-pure water and internal standard (deoxy-glucose
for monosaccharide and fucose for uronic acid analysis) was
added for HPAEC analysis. A large batch of each sample was
also depolymerized and distributed to the other participating
laboratories for analysis.

2.3.

Acid methanolysis

The dried plant materials (about 10 mg) were depolymerized


using acid methanolysis (Table 1) by the addition of 2 mL 2 M
HCl in anhydrous methanol (Sundberg et al., 1996). The samples were then kept at 100 C for either 5 h (ABO, Table 2) or 3 h
(KCL) before cooling to ambient temperature and neutralization by addition of pyridine. A calibration solution containing
equal amounts of the sugar monomers and uronic acids
(except 4-O-MeGlcA) (ABO) or double amounts of xylose, mannose, and glucose compared to the other ve sugars (KCL)
was also subjected to acid methanolysis under similar conditions. Additionally, 4 mL (ABO) or 1 mL (KCL) of methanol
solution containing the internal standard sorbitol (0.1 mg/mL)
was added to the samples. To exclude bers during analysis,
1 mL of clear solution was then transferred to a clean test tube
(ABO) or alternatively ltration of the sample with 0.45 m GHP
or Millipore lters was performed (KCL).

2.4.

Three different wood-derived samples and two samples from


annual plants were chosen for the current study. Norway
spruce thermomechanical pulp (spruce TMP) was obtained
from a Finnish pulp mill. A section of European aspen stemwood (aspen wood) was sampled from a fresh mature tree
growing in Southern Finland. The sample was splintered,
freeze-dried, and ground in a Wiley mill, producing particles
passing a 10-mesh screen. Both the spruce TMP and aspen
wood sample were Soxhlet extracted using acetone to remove
extractives, including possibly sugar monomers and dimers,
and phenolic glycosides. Bleached (TCF, totally chlorine-free
process) birch kraft pulp was obtained in dry sheet form from a
Finnish pulp mill. Wheat straw (Mykora, Finland) and oat spelt
(hulls) (Suomen Viljava, Finland) were freeze-dried and ground

573

Enzymatic hydrolysis

Total enzymatic hydrolysis (Table 1) described by Tenkanen


et al. (1995, 1999) was carried out to the kraft pulp sample. The enzyme preparation used was a combination of
commercial cellulase (Econase, lot 242039, AB Enzymes,
Finland), xylanase (Ecopulp HC, AB Enzymes, Finland), mannanase (Gamanase, Novozymes, Denmark) and b-glucosidase
(Novozyme 188, Novozymes, Denmark). The preparations
were combined and puried from contaminating sugars
and small molecular weight compounds by gel ltration.
The enzyme activities of the mixture were determined as
described earlier (Tenkanen et al., 1995, 1999). The mixture
applied for the hydrolysis was dosed by adding the preparation according to the target activities per gram of pulp as
follows: 50 FPU/g, 500010,000 nkat/g major endo-hydrolases
(endo-1,4-b-glucanase, endo-1,4-b-xylanase and endo-1,4-b-

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i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

mannanase), 10002000 nkat/g b-glucosidase, 100500 nkat/g


a-arabinosidase and a-galactosidase and 50200 nkat/g bmannosidase. About 0.5 g sample was hydrolyzed for 48 h at
40 C and pH 5 (sodium acetate buffer). Hereafter the samples were boiled for 5 min to inactivate the enzymes and then
centrifuged.

2.5.

GC-FID and GC/MS analysis

Methanolysates, calibration samples, and hydrolysates (from


BFH, Table 2) were neutralized with pyridine and dried under
nitrogen, and additionally in a vacuum desiccator at 40 C for
20 min before silylation overnight at room temperature using
150 L HMDS (hexamethyl disilazane), 70 L TMCS (trimethyl
chlorosilane), and 150 L of pyridine (ABO). Alternatively, the
samples were neutralized with pyridine and evaporated in a
vacuum evaporator with bath temperature of 45 C for 10 min,
stored in a freezer until analysis and then dried in a vacuum
oven at 40 C for 1 h on the analysis day (KCL). The samples
were transferred with 80 L of pyridine to small glass bottles for GC/MS analysis. Silylation was performed by adding
250 L of N,O-bis(trimethyl silyl)-triuoroacetamide containing 5% TMCS and heating of the samples in a heating block at
70 C for 1 h.
The GC-FID parameters were essentially according to
Sundberg et al. (1996). About 1 L silylated sample was injected
in a split mode (260 C, split ratio 1:20) into a 25 m 0.20 mm
i.d. column coated with dimethyl polysiloxane (HP-1, Agilent
Technologies), the lm thickness being 0.11 m. Alternatively,
a similar (5%-phenyl)-methyl polysiloxane coated column (HP5, Agilent Technologies) was used for comparison. The column
temperature was 100 C4 C/min180 C, 12 C/min290 C.
The FID detector temperature was 300 C. Hydrogen was used
as carrier gas at a ow rate of 0.8 mL/min. The PerkinElmer
TotalChrom (Version 6.2.1) Microsoft Windows -based software package was used for data acquisition. Calibration
factors were determined from the calibration samples after
methanolysis by the ratio of the total area of the different
sugar unit peaks to the area of the sorbitol peak. The calibration factor for 4-O-MeGlcA, which is not commercially
available in pure form, was assumed to be the same as for
GlcA.
The GC/MS parameters were according to Laine et al.
(2002). About 1 L silylated sample was injected via a split
injector (260 C, split ratio 1:50) into a 30 m 0.25 mm i.d.
column coated with (5%-phenyl)-methyl polysiloxane (HP5, Agilent Technologies), the lm thickness being 0.25 m.
The column temperature was 100 C (2 min)4 C/min220 C,
15 C/min300 C. Helium was used as carrier gas at a ow
rate of 1 mL/min. The detector conditions were 70 eV and
40600 amu. Quantication was subsequently done using relative molar response factors for each sugar (Saeman et al.,
1954).

2.6.

HPAEC-PAD analysis

The high-performance anion-exchange chromatography


analysis of monosaccharides and uronic acids was performed by using a CarboPac PA-1 column (guard column
4 50 mm and analytical column 4 250 mm) or PA-10

column (guard column 4 50 mm and analytical column


4 250 mm) (Dionex, Sunnyvale, CA, USA) (KCL) coupled to
Dionex DX 500 series chromatograph equipped with pulse
amperometric detection (Dionex ED 40) (Hausalo, 1995). For
the monosaccharide analysis the system was equilibrated
with 15 mM sodium hydroxide. After sample injection, 15 mM
sodium hydroxide was run through the column for 2 min and
from 2 to 36 min 100% of ultra-pure water was run isocratically
(VTT). Alternatively, only ultra-pure water was used as eluent
(KCL). A solution of 300 mM NaOH was added to the column
efuent before the PAD cell at a ow rate of 0.8 mL/min. The
column was washed with 100 mM sodium hydroxide/300 mM
sodium acetate for 3 min and then with 300 mM sodium
hydroxide for 4 min (VTT), or with 200 mM for 15 min (KCL).
For the uronic acid analysis the system was equilibrated with
100 mM sodium hydroxide. After injection, linear gradient
from 100 mM sodium hydroxide to 100 mM/300 mM sodium
acetate was run in 20 min. The column wash was identical
to that for the monosaccharide gradient. The ow rate was
1 mL/min, column temperature 30 C, sample temperature
15 C and injection volume 20 L (VTT), or sample temperature room temperature and injection volume 25 l (KCL). Data
were processed with Chromeleon software. The coefcient of
variation of the HPAEC analysis was less than 5%.

2.7.

HPAEC-Borate analysis

Wood sugars were separated on a 6.6 mm bore column of


115 mm length (Omnit) lled with the strong anion exchange
resin (MCI Gel CA08F (Mitsubishi) at 60 C). The mobile phase
(0.7 ml min1 ) was made of A: 0.3 M potassium borate buffer
pH 9.2 and B: 0.9 M potassium borate buffer pH 9.2. After
sample injection the separation was started with 90%A and
10%B. A linear gradient was run within 35 min to 10%A and
90%B. Data acquisition was stopped after 47 min. Sugar quantication was achieved by after-column derivatization with
Cu-bichinconinate (0.35 ml min1 ) at 105 C in a 30 m crocheted Teon coil of 0.3 mm inner diameter and detection
at 560 nm. Data were processed with the Dionex Chromeleon
software.

3.

Results and discussion

The aim of this work was to critically compare methods for carbohydrate analysis of solid plant materials with
uronic acid-containing polysaccharides used in analytical
laboratories today. The comparison was initiated by choosing ve different plant materials (oat spelt, wheat straw,
spruce thermomechanical pulp, aspen stemwood, and totally
chlorine-free (TCF) bleached hardwood kraft pulp) that represent various matrices for the polysaccharide-containing
material. Each participating laboratory was then asked to use
their in-house analytical method to determine the carbohydrate amount and composition (Tables 1 and 2). Both acid
hydrolysis and acid methanolysis were applied for all samples,
while enzymatic hydrolysis only was applied for the delignied TCF pulp. The subsequent analytical procedures were
GC-FID, using both an unpolar HP-1 and a semi-polar HP-5 column, GCMS, using an HP-5 column, an HPAEC-PAD method,

575

i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

Table 3 Carbohydrate composition, expressed as mg monosaccharide/100 mg dry substance, in the ve plant samples
determined using different depolymerization and analytical methods. Each sugar amount is the average of ve
determinations, except for the VTT and ABO analyses (duplicates) of the BFH hydrolysates (one hydrolysis only).
mg/100 mg dry substance
Ara

Rha

Xyl

Man

Gal

Glc

Oat spelt
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC
KCL methanolysis + GC

3.2
2.8
3.0
2.8
3.1
5.3
3.5

<0.1
<0.1
0.2
<0.1
<0.1
0.1
<0.1

28.2
30.0
28.7
29.0
29.4
36.6
22.2

<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1

1.4
1.3
1.3
1.3
1.3
1.5
1.8

33.8
32.0
35.4
34.5
33.9
3.5
4.2

Wheat straw
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC
KCL methanolysis + GC

2.4
2.2
2.5
2.2
2.4
3.4
3.9

0.1
<0.1
0.2
0.1
0.1
0.2
0.1

21.0
23.0
21.8
22.6
22.3
24.4
26.3

0.4
0.4
0.5
0.5
0.5
0.3
0.4

0.8
0.8
0.8
0.7
0.7
1.1
1.2

Spruce TMP
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC
KCL methanolysis + GC

1.2
1.2
1.1
1.1
1.2
1.7
1.1

0.1
0.1
0.1
0.1
0.2
0.3
0.2

5.0
5.6
5.4
5.5
5.6
6.6
4.8

11.8
13.0
12.8
12.7
11.9
12.6
9.3

Aspen stemwood
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC

0.3
0.3
0.3
0.3
0.4
0.5

0.3
0.3
0.4
0.3
0.4
0.5

16.5
18.0
17.1
17.6
18.1
21.8

<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1

<0.1
<0.1
0.1
<0.1
<0.1
<0.1
<0.1

23.8
25.0
23.5
24.0
24.4
25.0
17.7

Bleached birch kraft pulp


KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT enzymatic hydrolysis + HPAEC-PAD
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC

4-O-MeGlcA

GlcA

GalA

Sum

nd
0.2
0.1
0.4
<0.1
0.5
0.3

nd
0.3
nd
0.4
<0.1
0.8
0.7

nd
0.1
nd
0.2
<0.1
0.5
0.3

66.6
66.7
68.7
68.6
67.7
48.8
33.0

39.1
42.0
40.9
40.7
39.6
6.2
6.3

nd
0.4
0.3
0.7
<0.1
0.9
0.7

nd
0.1
nd
0.2
<0.1
0.6
0.3

nd
0.2
nd
0.3
<0.1
0.6
0.5

63.9
69.1
67.0
68.0
65.6
37.6
39.7

1.9
1.9
1.9
1.8
1.7
2.5
2.5

44.0
49.0
48.3
47.3
44.2
5.5
5.7

nd
0.4
0.6
1.0
<0.1
1.3
0.9

nd
<0.1
nd
<0.1
<0.1
0.2
0.1

nd
0.6
nd
0.8
<0.1
1.9
1.4

64.0
71.9
70.2
70.3
64.8
32.7
26.1

2.1
2.2
2.4
2.0
2.1
1.8

0.6
0.5
0.6
0.5
0.5
0.8

46.5
51.0
49.6
48.0
47.7
3.3

nd
0.8
1.0
1.5
<0.1
2.2

nd
<0.1
nd
<0.1
<0.1
0.3

nd
0.9
nd
1.0
<0.1
2.8

66.4
73.9
71.4
71.2
69.1
34.1

0.5
0.5
0.6
0.4
0.5
0.5
0.3

<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.2

71.8
80.0
74.9
72.0
72.6
72.6
4.4

nd
0.2
0.1
0.3
0.4
<0.1
0.3

nd
<0.1
nd
<0.1
<0.1
<0.1
<0.1

nd
<0.1
nd
<0.1
0.1
<0.1
<0.1

96.1
105.7
99.2
96.7
97.9
98.1
22.8

nd = not determined.

and an HPAEC-Borate method. Furthermore, the possibility


of analyzing methanolysates using the HPAEC-PAD system
was tested. The chosen approach did not consent to recognizing the inuence of different steps in the method, but rather
allowed nding a convenient method, or methods, that would
give the best results; i.e. the largest total amount of each neutral or acidic sugar unit.

3.1.

Carbohydrate amount and composition

The carbohydrate amount and composition of all ve samples determined using the different methods are presented
in Table 3. Note that to obtain the approximate amount
of polysaccharides present in the samples, the monosaccharide amounts in Table 3 should be multiplied by 0.9 to

account for the loss of a water molecule in the polysaccharide. Neither the amount of acetyl groups nor the amounts
of methyl groups (or other functional groups) were determined, so these will be lacking from the tentative amounts
of polysaccharides. The oat spelt and wheat straw samples
were not pre-extracted and the results may thus contain small
amounts of sugars from, for example, phenolic glycosides. It
should also be pointed out that the acid methanolysis method
does not manage to cleave highly ordered (or crystalline)
polysaccharides and thus the amount of glucose represent
only glucose in hemicelluloses or very amorphous parts of
cellulose. A somewhat smaller amount of mannose in several
samples for the acid methanolysis-GC method compared to
the other methods probably arrives from the fact that part of
the galactoglucomannans present in spruce may be deacety-

576

i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

Table 4 Carbohydrate composition, expressed as mole% of the total sugar amount given in Table 3 in the ve plant
samples determined using different depolymerization and analytical methods.
mole% of total sugar amount
Ara

Rha

Xyl

Man

Gal

Glc

4-O-MeGlcA

GlcA

GalA

Oat spelt
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC
KCL methanolysis + GC

5.3
4.6
4.8
4.5
5.1
11.2
11.1

<1
<1
<1
<1
<1
<1
<1

46.4
49.2
45.9
46.5
47.5
76.9
70.1

<1
<1
<1
<1
<1
<1
<1

1.9
1.8
1.7
1.7
1.7
2.7
4.8

46.3
43.7
47.2
46.1
45.7
6.2
11.1

nd
<1
<1
<1
<1
<1
<1

nd
<1
nd
<1
<1
1.3
1.6

nd
<1
nd
<1
<1
<1
<1

Wheat straw
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC
KCL methanolysis + GC

4.2
3.6
4.2
3.6
4.0
9.5
10.2

<1
<1
<1
<1
<1
<1
<1

36.7
37.2
36.4
37.2
38.0
68.2
69.2

<1
<1
<1
<1
<1
<1
<1

1.1
1.1
1.1
1.0
1.1
2.5
2.6

57.1
56.7
56.9
55.9
56.1
14.3
13.8

nd
<1
<1
<1
<1
1.7
1.4

nd
<1
nd
<1
<1
1.2
<1

nd
<1
nd
<1
<1
1.4
1.0

Spruce TMP
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC
KCL methanolysis + GC

2.2
2.0
1.8
1.8
2.2
6.1
5.0

<1
<1
<1
<1
<1
<1
<1

9.3
9.2
9.1
9.2
10.2
23.4
21.5

18.1
17.8
17.9
17.8
17.9
37.0
34.3

2.9
2.6
2.7
2.5
2.5
7.4
9.3

67.3
67.0
67.6
66.2
66.8
16.3
21.2

nd
<1
<1
1.2
<1
3.4
2.9

nd
<1
nd
<1
<1
<1
<1

nd
<1
nd
1.1
<1
5.1
4.9

Aspen stemwood
KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC

<1
<1
<1
<1
<1
1.5

<1
<1
<1
<1
<1
1.6

28.4
27.9
27.4
28.4
29.8
68.8

3.0
2.8
3.2
2.7
2.9
4.8

<1
<1
<1
<1
<1
2.0

66.7
65.8
66.3
64.4
65.5
8.7

nd
<1
1.2
1.7
<1
4.9

nd
<1
nd
<1
<1
<1

nd
1.1
nd
1.3
<1
6.9

Bleached birch kraft pulp


KCL hydrolysis + HPAEC-PAD
VTT hydrolysis + HPAEC-PAD
BFH hydrolysis + HPAEC-Borate
VTT enzymatic hydrolysis + HPAEC-PAD
VTT HPAEC-PAD on BFH hydrolysates
ABO GC on BFH hydrolysates
ABO methanolysis + GC

<1
<1
<1
<1
<1
<1
<1

<1
<1
<1
<1
<1
<1
<1

24.8
23.6
23.7
24.8
24.9
24.8
77.5

<1
<1
<1
<1
<1
<1
1.1

<1
<1
<1
<1
<1
<1
<1

75.2
75.7
75.5
74.5
74.1
74.5
19.5

nd
<1
<1
<1
<1
<1
1.2

nd
<1
nd
<1
<1
<1
<1

nd
<1
nd
<1
<1
<1
<1

nd = not determined.

lated, re-deposited, and thus highly ordered (or crystalline)


on free cellulose surfaces in the bers. However, this kind
of selectivity can in certain cases give additional information. For example, the difference between the xylose amount
in the kraft pulp sample determined by acid hydrolysis and
methanolysis represents the highly ordered (or crystalline)
part of re-adsorbed xylans on the surface.
Table 4 shows the results from Table 3 recalculated to
give the relative carbohydrate composition in the samples. It
has been suggested that presenting the relative carbohydrate
composition rather than the absolute one evens out variations due to instrument calibration (Jacobs, 2003; Jacobs et al.,
2003). However, the absolute composition given in Table 3 is
already quite satisfactory. Only the amount of glucose deter-

mined by HPAEC-PAD after hydrolysis at VTT (see Table 2)


(90.0 mg/100 mg for birch kraft pulp) may raise some questions, since that leads to a total polysaccharide amount above
100%. It is evident that for analysis of neutral non-cellulose
sugars any of the tested methods should be adequate enough
to use.
The standard deviation for the amount of individual sugars
(ve determinations) was less than 5% for the major sugars in
a sample (not shown), while it could be higher for the minor
sugars (from a few percent to 20%). Similar results concerning
the standard deviation were also obtained in an earlier study
on carbohydrate analysis by GC and HPLC after acid or enzymatic hydrolysis of pulp samples (Jacobs, 2003; Jacobs et al.,
2003).

i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

3.2.

Analysis of uronic acids

As expected, acid methanolysis combined with GC analysis


gave the largest amounts of uronic acids (Table 3). Obviously,
the absence of uronic acids in the BFH hydrolysates analyzed
at ABO is caused by the fact that the hydrolysates were stored
for several weeks before neutralization, which caused degradation of the labile acids. The analysis of uronic acids (except
4-O-MeGlcA) using the HPAEC-PAD system usually requires
another elution prole (longer analysis time) and the analysis
of both neutral and acidic sugars in one single run is inconvenient. Therefore only one laboratory (VTT) analyzed the uronic
acids with HPAEC-PAD after acid hydrolysis.
De Ruiter et al. (1992) showed that both neutral and acidic
sugars can be analyzed in one single run using the HPAECPAD system (De Ruiter et al., 1992). Unfortunately, methyl
glycosides as such in methanolysates cannot be analyzed
using this system. Nevertheless, De Ruiter et al. (1992) showed
that applying hydrolysis with triuoroacetic acid (TFA) after
acid methanolysis sufciently converts methyl glycosides and
methyl ester methyl glycosides to free sugars, thus allowing
analysis by the HPAEC-PAD system. This approach was not
tested in the present work. Nevertheless, it is evident that the
use of acid methanolysis is to prefer over acid hydrolysis when
the total non-crystalline carbohydrates are analyzed.
No attempt was made to analyze the hexenuronic acid
(i.e. 4-deoxy-L-threo-hex-4-enopyranosyluronic acid) units
present in the bleached birch kraft pulp, since this was the
only sample where these acids should exist. Different methods for the analyses of hexenuronic acids are available, but
these may require additional analytical steps (Tenkanen et al.,
1995, 1999; Gellerstedt and Li, 1996; Dahlman et al., 2000; Li et
al., 2007).

3.3.

Depolymerization

The recommended standard method for acid hydrolysis of


extractive-free wood and pulp comprises a two-step hydrolysis using sulfuric acid (Tappi T 249 cm-85, 1985). The method
recommends the use of a calibration so that a monosaccharide solution containing the neutral sugars also converts
through the hydrolysis steps. Puls (1993) also discussed the
use of a calibration step in the hydrolysis. However, the prehydrolysis step releases mainly oligosaccharides from the
lignocellulosic matrix and the efcacy of this step is deeply
dependent on the matrix. The following post-hydrolysis step
mainly cleaves the bonds between sugar molecules in the
released oligosaccharides, so there is only little sense in calibrating with monosaccharides. The use of oligosaccharides
for calibration would be more realistic, but unfortunately such
well-dened, pure, and suitable products are not yet commercially available. Hence, many laboratories today use the acid
hydrolysis method without a calibration mixture. More important would be the optimization of the actual hydrolysis time
and temperature used for different types of samples, both in
the pre- and post-hydrolysis steps (Puls, 1993; Jacobs, 2003;
Jacobs et al., 2003). Puls (1993) comprehensively demonstrated
the effect of post-hydrolysis time on the sugar yields from two
tree species. However, optimizing the conditions for all different types of samples is tedious and for the present work the

577

aim was to study the general in-house methods capability to


perform for different plant materials.
Acid methanolysis is done in one step and a longer
treatment time is needed for plant materials compared to dissolved polysaccharides to allow a complete diffusion of sugar
monomer residues out of the lignocellulosic matrix (Sundberg
et al., 1996; Bertaud et al., 2002). The use of a calibration mixture allows for some correction of degradation also in the
methanolysis step, but it is not possible to account for all
differences in degradation of the released sugars due to the lignocellulosic matrix. However, it is reasonable to assume that
the stability of released sugars is quite high at the experimental conditions (2 M HCL in water-free MeOH), especially since
the acidity of the solution drops rather quickly (Chambers and
Clamp, 1971; Bertaud et al., 2002). The methanolysis method
is known for its good performance in cleaving glucuronosyl
bonds and little degradation of the uronic acids released. Nevertheless, highly ordered (crystalline) structures will not be
fully depolymerized with acid methanolysis, which is seen
in somewhat small amounts of mannose in most samples
and xylose in the bleached birch kraft pulp sample (Table 3).
The reason why the KCL methanolysis gave smaller amounts,
compared to the ABO methanolysis, for several sugars in the
oat and wheat samples is not fully understood, but one explanation may be the shorter treatment time of 3 h compared to
the 5 h treatment at ABO. The results for the wheat straw sample were comparable even with the different hydrolysis times
implying that the wheat straw sample depolymerized more
easily than the oat spelt or spruce TMP sample.

3.4.

GC analysis, using HP-1 and HP-5 columns

One minor drawback with GC is that the sugar monomer


residues need derivatization, preferably silylation, prior to
the analysis to render them volatile enough, which is one
additional time-consuming step. However, the possibility to
analyze both neutral and acidic carbohydrates in one single
run together with the excellent separation obtained especially
with the unpolar HP-1 column and the modern, highly automated, GC instruments overweighs the disadvantages without
difculty (Figs. 1 and 2). The use of hydrogen as carrier gas
instead of the more commonly used helium also gives slightly
better separation and thus less overlapping peaks (not shown).
Additionally, the introduction of modern hydrogen generators
has increased the safety in the laboratory. For comparison, all
samples were run also on an HP-5 column (Fig. 1). The HP-1
column should be preferred for analysis of the basic sugars in
this work, since there are more overlapping peaks on the HP-5
column. However, the combination of GC/MS, helium as carrier gas, and an HP-5 column also gave satisfactory results (not
shown). Nonetheless, for routine analyses, the use of GC/MS is
not necessary. In difcult cases though, the possibility to peak
identication with mass spectra is an advantage.
Methanolysis of a sugar leads to the formation of several
isomers due to well-known anomerization and ring isomerization processes (Bleton et al., 1996; Mejanelle et al., 2002).
The number of peaks, their retention times, and relative proportions are characteristic for each monosaccharide under the
same methanolysis conditions (Fig. 1). The peak pattern facilitates identication and anomalies can be identied when

578

i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

Fig. 1 GC analysis of equal amounts of eight different sugars (calibration mixture) after methanolysis and silylation. Both
an HP-1 and an HP-5 column are here used for comparison.

the regular pattern is disturbed. The complexity of the chromatogram is not a problem to evaluate with modern software
programs.

3.5.

HPAEC-PAD analysis

High-performance anion-exchange chromatography (HPAEC)


using polymer-based stationary phases and high pH in combination with pulsed amperometric detection (PAD) allows
direct quantication of underivatized carbohydrates. Elution
at high pH allows the separation of carbohydrates as their
oxyanions. According to pKa values of the neutral monosaccharides in the order of 1214, they act as weak acids. At high

pH, they are partially ionized and strong anion exchange stationary phase can be used for separation. Gold electrodes are
used for PAD and in most instances the pH value of 0.1 M
sodium hydroxide is high enough (Huber and Bonn, 1995).
For the separations that are not performed under adequately
alkaline conditions, the detection requirement of high pH
can be satised by post column addition of alkaline reagents
(LaCourse, 1997). CarboPac PA1 column is used in mono-, oligoand polysaccharide analyses. Monosaccharides and uronic
acids are usually analyzed separately, because of faster gradient and runtime. Monosaccharides elute with a short 15 mM
sodium hydroxide pulse between 10 and 27 min with ultrapure water (Fig. 3). Uronic acids elute with 100 mM sodium
hydroxide/300 mM sodium acetate gradient in 15 min.

3.6.

Fig. 2 GC-FID analysis of a spruce TMP sample, after


methanolysis and silylation, using an HP-1 column.

HPAEC-Borate analysis

Borate complex anion exchange chromatography using Cubicinchoninate for detection also allows direct quantication
of underivatized sugars. Whereas in HPAEC-PAD a weak anion
exchange resin is used, HPAEC-Borate requires a strong anion
exchange gel as stationary phase. Unfortunately there seems
to be only one manufacturer of the required HPLC-grade
resin left so that certain expenditure is required for a constant supply. Applying a linear gradient between 0.3 M and
0.9 M potassium borate buffer pH 9.2 allows the separation of
the wood sugars as their borate complexes. Due to the high
borate concentration, the column continuously regenerates
itself so that acid hydrolysates can be directly injected without neutralization. A Cu-bicinchoninate reagent is added to

i n d u s t r i a l c r o p s a n d p r o d u c t s 2 9 ( 2 0 0 9 ) 571580

579

samples. However, acid and enzymatic hydrolysis gives the


highest recoveries for bleached chemical pulp samples. Acid
hydrolysis is also required for crystalline polysaccharides,
but the strong acid conditions clearly lead to degradation of
labile sugars. For analysis of the total amount of sugar units,
hence including cellulose, other non-crystalline hemicelluloses, and pectins, a combination of the methanolysis and
hydrolysis methods is recommended. Furthermore, time and
money allowing, special emphasis should be put into calibrating the system of choice for each new plant material to
analyze.

Acknowledgements

Fig. 3 HPAEC-PAD chromatograms of the spruce TMP


hydrolysate and the carbohydrate standard solution used
for calibration. Dotted line = spruce TMP.

Prof. Maija Tenkanen is acknowledged for discussions and


advice. This work is part of the activities within the European Polysaccharide Network of Excellence (EPNOE) and also
within the COST E41 and COST D29 actions. This work is also
part of the activities within the Finnish Centre of Excellence
Programme (20002011) by the Academy of Finland.

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Fig. 4 HPAEC-Borate chromatogram of the spruce TMP


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including 4-O-Me-glucuronic acid elute within 50 min (Fig. 4).

4.

Conclusions

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