Professional Documents
Culture Documents
available at www.sciencedirect.com
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-
merization of ve different plant materials containing uronic acids. The analyzed plant
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
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
1.
Introduction
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
572
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
Internal standard
Reaction
conditions
Calibration
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)
Except 4-O-MeGlcA.
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
c
d
BFHb
e
KCLc
ABOd
2.
Experimental
2.1.
Materials
2.2.
Acid hydrolysis
2.3.
Acid methanolysis
2.4.
573
Enzymatic hydrolysis
574
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
2.5.
2.6.
HPAEC-PAD analysis
2.7.
HPAEC-Borate analysis
3.
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
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.
3.1.
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
<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.
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.
3.3.
Depolymerization
577
3.4.
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
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.
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
Acknowledgements
references
4.
Conclusions
Acid hydrolysis, acid methanolysis, and enzymatic hydrolysis have been compared for depolymerization of ve different
plant materials containing uronic acids. GC (using both HP1 and HP-5 capillary columns and FID and MSD detectors),
HPAEC-PAD, and HPAEC-Borate techniques were compared for
the subsequent analysis of the released monosaccharides.
Acid methanolysis combined with GC analysis is a convenient method for obtaining the sugar unit composition and
amount of non-crystalline polysaccharides in different plant
materials. The methanolysis method is generally superior to
the hydrolysis method for xylan- and uronic acid-containing
580
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